Structured Review

BioLegend anti igd pe
Expression of ST6Gal-1, α2,6-sialyl ligands, and CD22 in B cells. (A) Bone marrow immature (IM), <t>IgM-high,</t> and mature (BMM), as well as splenic <t>IgD-/CD21-,</t> IgD+/CD21+, marginal zone (MZ), and follicular (FO) populations were isolated by fluorescence activated cell sorting (FACS) ( > 94% purity). RT-qPCR was performed for ST6Gal-1 transcripts, and representative results of three independent experiments shown relative to β2-microglobulin ( n = 3). Western blot analysis of protein levels in splenic populations is quantified relative to β-actin ( n = 3). (B) Mean SNA reactivity is shown for bone marrow and splenic B cell subsets ( n = 5 or 10). (C) Frequency of cell surface CD22 expression in BM and splenic B cell populations ( n = 5). (D) Relative RNA expression of ST6Gal-1 and SNA reactivity are compared, with standard deviations shown in both dimensions, and arrows indicating select developmental steps. (E) CD22 expression and SNA reactivity is compared, with standard deviations of measurement shown in both dimensions. Arrows indicate sequence of B cell development.
Anti Igd Pe, supplied by BioLegend, used in various techniques. Bioz Stars score: 91/100, based on 2056 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti igd pe/product/BioLegend
Average 91 stars, based on 2056 article reviews
Price from $9.99 to $1999.99
anti igd pe - by Bioz Stars, 2020-09
91/100 stars

Images

1) Product Images from "Systemic ST6Gal-1 Is a Pro-survival Factor for Murine Transitional B Cells"

Article Title: Systemic ST6Gal-1 Is a Pro-survival Factor for Murine Transitional B Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02150

Expression of ST6Gal-1, α2,6-sialyl ligands, and CD22 in B cells. (A) Bone marrow immature (IM), IgM-high, and mature (BMM), as well as splenic IgD-/CD21-, IgD+/CD21+, marginal zone (MZ), and follicular (FO) populations were isolated by fluorescence activated cell sorting (FACS) ( > 94% purity). RT-qPCR was performed for ST6Gal-1 transcripts, and representative results of three independent experiments shown relative to β2-microglobulin ( n = 3). Western blot analysis of protein levels in splenic populations is quantified relative to β-actin ( n = 3). (B) Mean SNA reactivity is shown for bone marrow and splenic B cell subsets ( n = 5 or 10). (C) Frequency of cell surface CD22 expression in BM and splenic B cell populations ( n = 5). (D) Relative RNA expression of ST6Gal-1 and SNA reactivity are compared, with standard deviations shown in both dimensions, and arrows indicating select developmental steps. (E) CD22 expression and SNA reactivity is compared, with standard deviations of measurement shown in both dimensions. Arrows indicate sequence of B cell development.
Figure Legend Snippet: Expression of ST6Gal-1, α2,6-sialyl ligands, and CD22 in B cells. (A) Bone marrow immature (IM), IgM-high, and mature (BMM), as well as splenic IgD-/CD21-, IgD+/CD21+, marginal zone (MZ), and follicular (FO) populations were isolated by fluorescence activated cell sorting (FACS) ( > 94% purity). RT-qPCR was performed for ST6Gal-1 transcripts, and representative results of three independent experiments shown relative to β2-microglobulin ( n = 3). Western blot analysis of protein levels in splenic populations is quantified relative to β-actin ( n = 3). (B) Mean SNA reactivity is shown for bone marrow and splenic B cell subsets ( n = 5 or 10). (C) Frequency of cell surface CD22 expression in BM and splenic B cell populations ( n = 5). (D) Relative RNA expression of ST6Gal-1 and SNA reactivity are compared, with standard deviations shown in both dimensions, and arrows indicating select developmental steps. (E) CD22 expression and SNA reactivity is compared, with standard deviations of measurement shown in both dimensions. Arrows indicate sequence of B cell development.

Techniques Used: Expressing, Isolation, Fluorescence, FACS, Quantitative RT-PCR, Western Blot, RNA Expression, Sequencing

Cell non-autonomous ST6Gal-1 influences sialylation and abundance of early transitional B cell populations. CD45.1+ whole bone marrow cells from wild-type or St6gal1-KO mice were adoptively transferred to irradiated CD45.2+ hosts. Mice were allowed to recover for 6 weeks before analysis of bone marrow and splenic B cells. (A) SNA reactivity of bone marrow and splenic B cell subsets of CD45.1+ donor cells. (B) Frequencies of CD45.1+ IM, IgM-high, BMM, IgD-/CD21-, IgD+/CD21+, MZ, and FO B cells as a fraction of total CD45.1+ B cells ( n = 5). (C) Immunofluorescence microscopy staining anti-IgM (red) and anti-IgD (green) in chimeras. Splenic B cell populations indicated are identified accordingly - T1: IgM+/IgD-, extrafollicular; T2 and FO: IgM-variable/IgD+, follicular, MZ: IgM+/IgD-, marginal sinus. * P
Figure Legend Snippet: Cell non-autonomous ST6Gal-1 influences sialylation and abundance of early transitional B cell populations. CD45.1+ whole bone marrow cells from wild-type or St6gal1-KO mice were adoptively transferred to irradiated CD45.2+ hosts. Mice were allowed to recover for 6 weeks before analysis of bone marrow and splenic B cells. (A) SNA reactivity of bone marrow and splenic B cell subsets of CD45.1+ donor cells. (B) Frequencies of CD45.1+ IM, IgM-high, BMM, IgD-/CD21-, IgD+/CD21+, MZ, and FO B cells as a fraction of total CD45.1+ B cells ( n = 5). (C) Immunofluorescence microscopy staining anti-IgM (red) and anti-IgD (green) in chimeras. Splenic B cell populations indicated are identified accordingly - T1: IgM+/IgD-, extrafollicular; T2 and FO: IgM-variable/IgD+, follicular, MZ: IgM+/IgD-, marginal sinus. * P

Techniques Used: Mouse Assay, Irradiation, Immunofluorescence, Microscopy, Staining

2) Product Images from "TRPA1 inhibition ameliorates pressure overload-induced cardiac hypertrophy and fibrosis in mice"

Article Title: TRPA1 inhibition ameliorates pressure overload-induced cardiac hypertrophy and fibrosis in mice

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2018.08.022

TRPA1 inhibition reduces M2 macrophage polarization. (A) Immunofluorescence analysis of M2 macrophages (anti-CD206) in mice at 4 weeks after TAC surgery (n = 7). (B) Flow cytometry analysis of M2 macrophage (CD45 + F4/80 + CD206 + ) expression in mice at 4 weeks after TAC surgery (n = 4). (C) RT-PCR analysis of interleukin-4 (IL-4), IL-10 and transforming growth factor-β (TGF-β) expression in bone marrow–derived macrophages (BMDMs) cultured with and without Ang II (n = 8). *P
Figure Legend Snippet: TRPA1 inhibition reduces M2 macrophage polarization. (A) Immunofluorescence analysis of M2 macrophages (anti-CD206) in mice at 4 weeks after TAC surgery (n = 7). (B) Flow cytometry analysis of M2 macrophage (CD45 + F4/80 + CD206 + ) expression in mice at 4 weeks after TAC surgery (n = 4). (C) RT-PCR analysis of interleukin-4 (IL-4), IL-10 and transforming growth factor-β (TGF-β) expression in bone marrow–derived macrophages (BMDMs) cultured with and without Ang II (n = 8). *P

Techniques Used: Inhibition, Immunofluorescence, Mouse Assay, Flow Cytometry, Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Cell Culture

TRPA1 inhibition prevents pressure overload-induced macrophage infiltration in cardiac tissues. (A) Immunohistochemical analysis of CD3 and CD68 in heart sections (n = 7, scale bar, 50 μm). (B–C) Flow cytometry analysis of CD45 + cells and T cells (CD45 + CD3 + ) in mice at 4 weeks after TAC surgery (n = 4). *P
Figure Legend Snippet: TRPA1 inhibition prevents pressure overload-induced macrophage infiltration in cardiac tissues. (A) Immunohistochemical analysis of CD3 and CD68 in heart sections (n = 7, scale bar, 50 μm). (B–C) Flow cytometry analysis of CD45 + cells and T cells (CD45 + CD3 + ) in mice at 4 weeks after TAC surgery (n = 4). *P

Techniques Used: Inhibition, Immunohistochemistry, Flow Cytometry, Cytometry, Mouse Assay

3) Product Images from "Immune checkpoints PVR and PVRL2 are prognostic markers in AML and their blockade represents a new therapeutic option"

Article Title: Immune checkpoints PVR and PVRL2 are prognostic markers in AML and their blockade represents a new therapeutic option

Journal: Oncogene

doi: 10.1038/s41388-018-0288-y

Combined blocking of PVR and PVRL2 on primary AML blasts increases the cytotoxic effects of HD-PBMCs. Mononuclear cells containing at least 75% blasts from bone marrow aspirates of ten different, newly diagnosed AML patients were stained with CMFDA (CellTracker™), mixed with HD-PBMCs as effector cells, and incubated for 72 h. Blocking of PVR and PVRL2 alone could increase the specific lysis of primary blasts in four ( a , b , c , f ) of the ten analyzed patients, and augment the anti-leukemic effect of the BiTE® antibody construct AMG 330 in five of nine patients ( a – e ). The sample of patient ( f ) was CD33 negative, and therefore excluded from the AMG 330 experiment. Results are depicted as the mean of technical triplicates ± SD of dead target cells
Figure Legend Snippet: Combined blocking of PVR and PVRL2 on primary AML blasts increases the cytotoxic effects of HD-PBMCs. Mononuclear cells containing at least 75% blasts from bone marrow aspirates of ten different, newly diagnosed AML patients were stained with CMFDA (CellTracker™), mixed with HD-PBMCs as effector cells, and incubated for 72 h. Blocking of PVR and PVRL2 alone could increase the specific lysis of primary blasts in four ( a , b , c , f ) of the ten analyzed patients, and augment the anti-leukemic effect of the BiTE® antibody construct AMG 330 in five of nine patients ( a – e ). The sample of patient ( f ) was CD33 negative, and therefore excluded from the AMG 330 experiment. Results are depicted as the mean of technical triplicates ± SD of dead target cells

Techniques Used: Blocking Assay, Staining, Incubation, Lysis, Construct

PVR and PVRL2 double-knockout cells recapitulate antibody effects in vitro and prolong the survival of NSG mice reconstituted with human T cells in vivo. By using CRISPR/Cas9, a polyclonal population of MV4-11 harboring double-knockout cells of PVR and PVRL2 was generated. Either MV4-11 wildtype or double-knockout cells were incubated with HD-PBMCs ( a ) or CD3 + cells ( b ) for 24 h without or with AMG 330. For statistical analysis, Mann–Whitney U -tests were performed (# p ≤ 0.05; * p ≤ 0.001, n = 3). c Immunodeficient NSG mice were transplanted with either MV4-11 wildtype (WT) cells or PVR- and PVRL2-double-knockout (KO) cells and reconstituted with human T cells. Treatment consisted of daily intraperitoneal application of either placebo ( n = 13 for WT and n = 12 for KO) or 15 µg/kg AMG 330 ( n = 12 for WT and n = 15 for KO). Log-rank tests were performed: WT placebo vs. KO placebo p
Figure Legend Snippet: PVR and PVRL2 double-knockout cells recapitulate antibody effects in vitro and prolong the survival of NSG mice reconstituted with human T cells in vivo. By using CRISPR/Cas9, a polyclonal population of MV4-11 harboring double-knockout cells of PVR and PVRL2 was generated. Either MV4-11 wildtype or double-knockout cells were incubated with HD-PBMCs ( a ) or CD3 + cells ( b ) for 24 h without or with AMG 330. For statistical analysis, Mann–Whitney U -tests were performed (# p ≤ 0.05; * p ≤ 0.001, n = 3). c Immunodeficient NSG mice were transplanted with either MV4-11 wildtype (WT) cells or PVR- and PVRL2-double-knockout (KO) cells and reconstituted with human T cells. Treatment consisted of daily intraperitoneal application of either placebo ( n = 13 for WT and n = 12 for KO) or 15 µg/kg AMG 330 ( n = 12 for WT and n = 15 for KO). Log-rank tests were performed: WT placebo vs. KO placebo p

Techniques Used: Double Knockout, In Vitro, Mouse Assay, In Vivo, CRISPR, Generated, Incubation, MANN-WHITNEY

Impact of PVR and PVRL2 expression on clinical outcome. Microarray-based gene expression data of 290 AML patients (cohort B) were divided into low and high expressors and analyzed for OS. High expression of either PVR or PVRL2 correlated significantly with a shortened overall survival
Figure Legend Snippet: Impact of PVR and PVRL2 expression on clinical outcome. Microarray-based gene expression data of 290 AML patients (cohort B) were divided into low and high expressors and analyzed for OS. High expression of either PVR or PVRL2 correlated significantly with a shortened overall survival

Techniques Used: Expressing, Microarray

PVR and PVRL2 are highly expressed on AML cell lines and primary CD33 + AML blasts. PVR and PVRL2 protein expression, as depicted by the percentage of CD33 + cells as well as median fluorescence intensity as the measure of expression intensity on AML cell lines ( n = 9; a , b ) and CD33 + AML blasts from untreated patients ( n = 17; c , d ). Black dashes represent the median
Figure Legend Snippet: PVR and PVRL2 are highly expressed on AML cell lines and primary CD33 + AML blasts. PVR and PVRL2 protein expression, as depicted by the percentage of CD33 + cells as well as median fluorescence intensity as the measure of expression intensity on AML cell lines ( n = 9; a , b ) and CD33 + AML blasts from untreated patients ( n = 17; c , d ). Black dashes represent the median

Techniques Used: Expressing, Fluorescence

Blocking of the TIGIT-PVR/PVRL2 axis increases the lysis of AML cell lines. HD-PBMC-mediated lysis, with subject to the blocking of PVR and PVRLs on AML cell lines TF-1 ( a , n = 3), Molm-13 ( b , n = 6), Kasumi-1 ( c , n = 3), was measured after 24 h. The effect of blocking the receptor TIGIT on effector cells was examined for the cell lines TF-1 ( d , n = 5) and MV4-11 ( e , n = 3). Results are depicted as the mean ± SD fold changes (FC) of dead target cells, relative to the control without blocking antibodies. Measurements were performed in technical triplicates and for statistical analysis Mann–Whitney U -tests were performed (# p ≤ 0.05; * p ≤ 0.001)
Figure Legend Snippet: Blocking of the TIGIT-PVR/PVRL2 axis increases the lysis of AML cell lines. HD-PBMC-mediated lysis, with subject to the blocking of PVR and PVRLs on AML cell lines TF-1 ( a , n = 3), Molm-13 ( b , n = 6), Kasumi-1 ( c , n = 3), was measured after 24 h. The effect of blocking the receptor TIGIT on effector cells was examined for the cell lines TF-1 ( d , n = 5) and MV4-11 ( e , n = 3). Results are depicted as the mean ± SD fold changes (FC) of dead target cells, relative to the control without blocking antibodies. Measurements were performed in technical triplicates and for statistical analysis Mann–Whitney U -tests were performed (# p ≤ 0.05; * p ≤ 0.001)

Techniques Used: Blocking Assay, Lysis, MANN-WHITNEY

Blocking of the TIGIT-PVR/PVRL2 axis results in increased levels of Granzyme B secretion of immune cells. TF-1 ( a – d ) and MV4-11 ( e , f ) target cells were mixed with HD-PBMCs and incubated with blocking antibodies against PVR and PVRL2 or TIGIT in the presence or absence of the BiTE® antibody construct AMG 330. After 24 h, supernatants were harvested and human Granzyme B concentration was measured using ELISA. Results are depicted as the mean ± SD Granzyme B concentration of at least three independent experiments. For statistical analysis paired t -tests were performed (# p ≤ 0.05)
Figure Legend Snippet: Blocking of the TIGIT-PVR/PVRL2 axis results in increased levels of Granzyme B secretion of immune cells. TF-1 ( a – d ) and MV4-11 ( e , f ) target cells were mixed with HD-PBMCs and incubated with blocking antibodies against PVR and PVRL2 or TIGIT in the presence or absence of the BiTE® antibody construct AMG 330. After 24 h, supernatants were harvested and human Granzyme B concentration was measured using ELISA. Results are depicted as the mean ± SD Granzyme B concentration of at least three independent experiments. For statistical analysis paired t -tests were performed (# p ≤ 0.05)

Techniques Used: Blocking Assay, Incubation, Construct, Concentration Assay, Enzyme-linked Immunosorbent Assay

T-cell mediated lysis of the BiTE® antibody construct AMG 330 is significantly enhanced by additional administration of PVR and PVRL2 or TIGIT blocking antibodies. TF-1 ( a , n = 3), Molm-13 ( b , n = 6), Kasumi-1 ( c , n = 6) cells were incubated with HD-PBMCs and AMG 330 in the presence or absence of blocking antibodies against PVR or PVRL2. Blocking the receptor TIGIT on immune cells showed similar results for the cell line TF-1 ( d , n = 5) and MV4-11 ( e , n = 3). Results are depicted as the mean ± SD fold change (FC) of dead target cells, relative to the control without blocking antibodies. Lysis is mediated via CD3 + cells, as comparing HD-PBMCs and purified CD3 + cells from the same donor showed comparable results using the cell line TF-1 ( f , n = 2). Results are depicted as the mean ± SD of dead target cells of two independent experiments. Measurements were performed in technical triplicates, and for statistical analysis Mann–Whitney U -tests were performed (# p ≤ 0.05; * p ≤ 0.001)
Figure Legend Snippet: T-cell mediated lysis of the BiTE® antibody construct AMG 330 is significantly enhanced by additional administration of PVR and PVRL2 or TIGIT blocking antibodies. TF-1 ( a , n = 3), Molm-13 ( b , n = 6), Kasumi-1 ( c , n = 6) cells were incubated with HD-PBMCs and AMG 330 in the presence or absence of blocking antibodies against PVR or PVRL2. Blocking the receptor TIGIT on immune cells showed similar results for the cell line TF-1 ( d , n = 5) and MV4-11 ( e , n = 3). Results are depicted as the mean ± SD fold change (FC) of dead target cells, relative to the control without blocking antibodies. Lysis is mediated via CD3 + cells, as comparing HD-PBMCs and purified CD3 + cells from the same donor showed comparable results using the cell line TF-1 ( f , n = 2). Results are depicted as the mean ± SD of dead target cells of two independent experiments. Measurements were performed in technical triplicates, and for statistical analysis Mann–Whitney U -tests were performed (# p ≤ 0.05; * p ≤ 0.001)

Techniques Used: Lysis, Construct, Blocking Assay, Incubation, Purification, MANN-WHITNEY

4) Product Images from "Endothelial progenitor cell-conditioned medium promotes angiogenesis and is neuroprotective after spinal cord injury"

Article Title: Endothelial progenitor cell-conditioned medium promotes angiogenesis and is neuroprotective after spinal cord injury

Journal: Neural Regeneration Research

doi: 10.4103/1673-5374.232484

Effects of EPC-CM on inflammatory cytokine levels in vitro and in vivo . (A) Representative flow cytometry data showing the effect of EPC-CM on BMDMs. (a) Mature BMDMs were defined as CD11b + /F4/80 + subpopulations (upper right), with the purity displayed as percentage of the parent population. (b) Control BMDMs were incubated with CD11b and F4/80 antibody and used to set up the gate. (c) Control BMDMs without LPS stimulation were incubated with anti-rat CD11b, F4/80, CD86 and CD206 antibodies. M1 macrophages are CD11b + /F4/80 + /CD86 + /CD206 − (Q1), whereas M2 macrophages are CD11b + /F4/80 + /CD86 − /CD206 + (Q3). (d) BMDMs treated with LPS. (e) BMDMs cultured with Con-M and simultaneously stimulated with LPS. (f) BMDMs cultured with EPC-CM and stimulated with LPS. (B) Quantitation of M1 and M2 cells among the different groups. Compared with the Con-M group, EPC-CM significantly reduced M1 activation, while M2 cells remained relatively unchanged. (C) mRNA expression levels of inflammatory cytokines (optical density ratio) among groups. (D) Immunofluorescence staining for CD86 (M1 marker) and CD206 (M2 marker) in the epicenter 7 days after SCI ( n = 5 per group; green: CD86; red: CD206; blue: DAPI). (E) Quantification of CD86- and CD206-positive cells at 7 days after SCI. ** P
Figure Legend Snippet: Effects of EPC-CM on inflammatory cytokine levels in vitro and in vivo . (A) Representative flow cytometry data showing the effect of EPC-CM on BMDMs. (a) Mature BMDMs were defined as CD11b + /F4/80 + subpopulations (upper right), with the purity displayed as percentage of the parent population. (b) Control BMDMs were incubated with CD11b and F4/80 antibody and used to set up the gate. (c) Control BMDMs without LPS stimulation were incubated with anti-rat CD11b, F4/80, CD86 and CD206 antibodies. M1 macrophages are CD11b + /F4/80 + /CD86 + /CD206 − (Q1), whereas M2 macrophages are CD11b + /F4/80 + /CD86 − /CD206 + (Q3). (d) BMDMs treated with LPS. (e) BMDMs cultured with Con-M and simultaneously stimulated with LPS. (f) BMDMs cultured with EPC-CM and stimulated with LPS. (B) Quantitation of M1 and M2 cells among the different groups. Compared with the Con-M group, EPC-CM significantly reduced M1 activation, while M2 cells remained relatively unchanged. (C) mRNA expression levels of inflammatory cytokines (optical density ratio) among groups. (D) Immunofluorescence staining for CD86 (M1 marker) and CD206 (M2 marker) in the epicenter 7 days after SCI ( n = 5 per group; green: CD86; red: CD206; blue: DAPI). (E) Quantification of CD86- and CD206-positive cells at 7 days after SCI. ** P

Techniques Used: In Vitro, In Vivo, Flow Cytometry, Cytometry, Incubation, Cell Culture, Quantitation Assay, Activation Assay, Expressing, Immunofluorescence, Staining, Marker

5) Product Images from "Blimp1 Prevents Methylation of Foxp3 and Loss of Regulatory T Cell Identity at Sites of Inflammation"

Article Title: Blimp1 Prevents Methylation of Foxp3 and Loss of Regulatory T Cell Identity at Sites of Inflammation

Journal: Cell Reports

doi: 10.1016/j.celrep.2019.01.070

Blimp1 Governs Treg Cell Identity in CNS Treg Cells Mixed bone marrow chimeras (MBMCs) were generated by reconstituting Rag1 −/− hosts with (CD45.1, wild-type) and Blimp1 ΔFoxp3 (CD45.2) bone marrow at a ratio of 1:1. The mixed bone marrow chimeras were immunized with MOG(35-55) in CFA to induce EAE. The mice were analyzed at the peak of EAE. (A) Chimerism between wild-type and Blimp1 ΔFoxp3 cells in the live CD4 + gate of spleen and CNS mononuclear cells and analysis of Foxp3 expression by flow cytometry in control (wild-type) Treg cells and Blimp1 ΔFoxp3 Treg cells isolated from the mixed bone marrow chimeras at the peak of EAE. Data are representative and summarized from three biological replicates. Symbols depict individual mice (bars, mean ± SD). Student’s t test ( ∗ p
Figure Legend Snippet: Blimp1 Governs Treg Cell Identity in CNS Treg Cells Mixed bone marrow chimeras (MBMCs) were generated by reconstituting Rag1 −/− hosts with (CD45.1, wild-type) and Blimp1 ΔFoxp3 (CD45.2) bone marrow at a ratio of 1:1. The mixed bone marrow chimeras were immunized with MOG(35-55) in CFA to induce EAE. The mice were analyzed at the peak of EAE. (A) Chimerism between wild-type and Blimp1 ΔFoxp3 cells in the live CD4 + gate of spleen and CNS mononuclear cells and analysis of Foxp3 expression by flow cytometry in control (wild-type) Treg cells and Blimp1 ΔFoxp3 Treg cells isolated from the mixed bone marrow chimeras at the peak of EAE. Data are representative and summarized from three biological replicates. Symbols depict individual mice (bars, mean ± SD). Student’s t test ( ∗ p

Techniques Used: Generated, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Isolation

CNS Treg Cells are Stable and Express Blimp1 in Response to Proinflammatory Cytokines (A) Mononuclear cells were isolated from the CNS of EAE mice at the peak of disease and were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin to analyze the expression of IL-10, IL-17, and IFN-γ in Treg cells by flow cytometry. Mean of eight biological replicates ± SD, derived from three independent experiments. Symbols depict individual mice (bars, mean ± SD). (B) CD4 + Foxp3 + Treg cells were sorted from the CNS and spleen (SPL) of Foxp3 (GFP) reporter mice at the peak of EAE and subjected to RNA-seq. Principal-component analysis. (C) Foxp3 expression by intracellular staining of splenic Treg cells and CNS Treg cells at the peak of EAE. Mean of six biological replicates ± SD, derived from two independent experiments. Symbols depict individual mice (bars, mean ± SD), t test, p
Figure Legend Snippet: CNS Treg Cells are Stable and Express Blimp1 in Response to Proinflammatory Cytokines (A) Mononuclear cells were isolated from the CNS of EAE mice at the peak of disease and were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin to analyze the expression of IL-10, IL-17, and IFN-γ in Treg cells by flow cytometry. Mean of eight biological replicates ± SD, derived from three independent experiments. Symbols depict individual mice (bars, mean ± SD). (B) CD4 + Foxp3 + Treg cells were sorted from the CNS and spleen (SPL) of Foxp3 (GFP) reporter mice at the peak of EAE and subjected to RNA-seq. Principal-component analysis. (C) Foxp3 expression by intracellular staining of splenic Treg cells and CNS Treg cells at the peak of EAE. Mean of six biological replicates ± SD, derived from two independent experiments. Symbols depict individual mice (bars, mean ± SD), t test, p

Techniques Used: Isolation, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Derivative Assay, RNA Sequencing Assay, Staining

Lack of Blimp1 in Treg Cells Results in Instability of Treg Cells through Epigenetic Changes (A) Schematic scheme of experimental set up. (B and C) Total Treg cells, namely, both control Foxp3 Treg cells ( Foxp3 Cre ) (B) or wild-type (C) and Blimp1 ΔFoxp3 Tre cells together, were sorted from spleen and draining lymph node of immunized mixed bone marrow chimeras on day 8 post-immunization and were transferred into Rag1 −/− recipient mice along with congenically marked CD25 - CD44 − CD90.1 + naive conventional CD4 + T cells (Thy 1.1 Tconv). On day 1 post-transfer, the recipient mice were immunized with MOG(35-55) and CFA. Mononuclear cells were isolated from the spleen and draining lymph node for analysis 8 days post-immunization. (C) Ki67 staining in wild-type and Blimp1 ΔFoxp3 Treg cells re-isolated from the spleen of the secondary hosts. Representative of three independent biological replicates. (D) Foxp3 expression was analyzed by intracellular staining. Frequency and geomean of Foxp3 + cells within the transferred Foxp3 Cre control Treg cells and Blimp1 ΔFoxp3 Treg cells isolated from the spleen of secondary hosts. Cumulative data of five biological replicates derived from two independent experiments. Symbols depict individual mice (bars, mean ± SD). Student’s t test ( ∗ p
Figure Legend Snippet: Lack of Blimp1 in Treg Cells Results in Instability of Treg Cells through Epigenetic Changes (A) Schematic scheme of experimental set up. (B and C) Total Treg cells, namely, both control Foxp3 Treg cells ( Foxp3 Cre ) (B) or wild-type (C) and Blimp1 ΔFoxp3 Tre cells together, were sorted from spleen and draining lymph node of immunized mixed bone marrow chimeras on day 8 post-immunization and were transferred into Rag1 −/− recipient mice along with congenically marked CD25 - CD44 − CD90.1 + naive conventional CD4 + T cells (Thy 1.1 Tconv). On day 1 post-transfer, the recipient mice were immunized with MOG(35-55) and CFA. Mononuclear cells were isolated from the spleen and draining lymph node for analysis 8 days post-immunization. (C) Ki67 staining in wild-type and Blimp1 ΔFoxp3 Treg cells re-isolated from the spleen of the secondary hosts. Representative of three independent biological replicates. (D) Foxp3 expression was analyzed by intracellular staining. Frequency and geomean of Foxp3 + cells within the transferred Foxp3 Cre control Treg cells and Blimp1 ΔFoxp3 Treg cells isolated from the spleen of secondary hosts. Cumulative data of five biological replicates derived from two independent experiments. Symbols depict individual mice (bars, mean ± SD). Student’s t test ( ∗ p

Techniques Used: Mouse Assay, Isolation, Staining, Expressing, Derivative Assay

6) Product Images from "CCR4-dependent reduction in the number and suppressor function of CD4+Foxp3+ cells augments IFN-γ-mediated pulmonary inflammation and aggravates tuberculosis pathogenesis"

Article Title: CCR4-dependent reduction in the number and suppressor function of CD4+Foxp3+ cells augments IFN-γ-mediated pulmonary inflammation and aggravates tuberculosis pathogenesis

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1240-3

CCR4 regulates the suppressor function of regulatory T cells during M. tuberculosis infection. WT (white circles) and CCR4 −/− (black circles) mice were infected with M. tuberculosis as described in the Fig. 1 or left uninfected. At 70 days of infection, the spleens were collected. CD4 + CD25 − (effector) cells (1 × 10 5 ) purified from the spleens of uninfected WT mice were co-cultured with CD4 + CD25 + (regulatory) cells (0.25 × 10 5 ) from the spleens of uninfected or infected WT or CCR4 −/− mice. Co-cultures were stimulated with ConA and after 96 h, proliferation was assessed a . Foxp3 expression on CD4 + CD25 + cells b , c . Proliferation was assessed by the Ki-67 expression on CD4 + CD25 − cells d , e . As a positive control, CD4 + CD25 − cells were stimulated with ConA in the absence of CD4 + CD25 + cells (gray circles). Data from two reproduced experiments #1 and #2 ( n = 3–5) expressed as the mean ± SEM. Symbols represent individual animals and bars show the difference ( P
Figure Legend Snippet: CCR4 regulates the suppressor function of regulatory T cells during M. tuberculosis infection. WT (white circles) and CCR4 −/− (black circles) mice were infected with M. tuberculosis as described in the Fig. 1 or left uninfected. At 70 days of infection, the spleens were collected. CD4 + CD25 − (effector) cells (1 × 10 5 ) purified from the spleens of uninfected WT mice were co-cultured with CD4 + CD25 + (regulatory) cells (0.25 × 10 5 ) from the spleens of uninfected or infected WT or CCR4 −/− mice. Co-cultures were stimulated with ConA and after 96 h, proliferation was assessed a . Foxp3 expression on CD4 + CD25 + cells b , c . Proliferation was assessed by the Ki-67 expression on CD4 + CD25 − cells d , e . As a positive control, CD4 + CD25 − cells were stimulated with ConA in the absence of CD4 + CD25 + cells (gray circles). Data from two reproduced experiments #1 and #2 ( n = 3–5) expressed as the mean ± SEM. Symbols represent individual animals and bars show the difference ( P

Techniques Used: Infection, Mouse Assay, Purification, Cell Culture, Expressing, Positive Control

Proposed model of collected findings. CCR4 regulates the magnitude of pulmonary inflammation at the chronic phase of M. tuberculosis infection by a mechanism dependent on the balance in the ratio of CD4 + Foxp3 + regulatory T cells and CD4 + Th1 effector cells, as well as in the suppressor function of regulatory T cells. Consequently, CCR4 deficiency accentuates the susceptibility to infection by a mechanism dependent on exacerbated magnitude of Th1 cell-mediated pulmonary inflammation
Figure Legend Snippet: Proposed model of collected findings. CCR4 regulates the magnitude of pulmonary inflammation at the chronic phase of M. tuberculosis infection by a mechanism dependent on the balance in the ratio of CD4 + Foxp3 + regulatory T cells and CD4 + Th1 effector cells, as well as in the suppressor function of regulatory T cells. Consequently, CCR4 deficiency accentuates the susceptibility to infection by a mechanism dependent on exacerbated magnitude of Th1 cell-mediated pulmonary inflammation

Techniques Used: Infection

Absence of CCR4 exacerbates lung Th1 inflammation and bacterial spreading into the lungs. WT (white circles) and CCR4 −/− (black circles) mice were infected with M. tuberculosis as described in the Fig. 1 . At 70 days of infection, the lungs were collected. Frequency of lung CD4 + Tbet + , CD4 + RORγt + and CD4 + GATA-3 + cells a . IFN-γ and IL-17 levels in the lung homogenates b . Representative analysis of IFN-γ-, IL-4-, IL-17- and IL-10-producing CD4 + cells c . Frequency of IFN-γ-, IL-4-, IL-17- and IL-10-producing CD4 + cells d . CXCR3 e and CCR5 f gene expression in the lung homogenates. Representative Ziehl-Neelsen staining on lung sections at 70 days of infection (magnification, ×400) g . Lung bacterial spreading score h . Data are representative of three-independent experiments ( n = 9–22) expressed as the mean ± SEM. Symbols represent individual animals and bars show the difference ( P
Figure Legend Snippet: Absence of CCR4 exacerbates lung Th1 inflammation and bacterial spreading into the lungs. WT (white circles) and CCR4 −/− (black circles) mice were infected with M. tuberculosis as described in the Fig. 1 . At 70 days of infection, the lungs were collected. Frequency of lung CD4 + Tbet + , CD4 + RORγt + and CD4 + GATA-3 + cells a . IFN-γ and IL-17 levels in the lung homogenates b . Representative analysis of IFN-γ-, IL-4-, IL-17- and IL-10-producing CD4 + cells c . Frequency of IFN-γ-, IL-4-, IL-17- and IL-10-producing CD4 + cells d . CXCR3 e and CCR5 f gene expression in the lung homogenates. Representative Ziehl-Neelsen staining on lung sections at 70 days of infection (magnification, ×400) g . Lung bacterial spreading score h . Data are representative of three-independent experiments ( n = 9–22) expressed as the mean ± SEM. Symbols represent individual animals and bars show the difference ( P

Techniques Used: Mouse Assay, Infection, Expressing, Staining

CCR4 increases lung CD4 + Foxp3 + cells during M. tuberculosis infection. WT (white circles) and CCR4 −/− (black circles) mice were infected with M. tuberculosis as described in the Fig. 1 . At 15, 30 and 70 days of infection (dpi) the lungs were collected. Representative analysis of CD4 + and CD4 + Foxp3 + cells a . Frequency and total number of CD4 + cells b , c and CD4 + Foxp3 + cells d , e . Data are representative of three-independent experiments ( n = 16–22), expressed as the mean ± SEM. Ratio of frequency f and total number g of CD4 + and CD4 + Foxp3 + cells obtained from lungs of infected WT and CCR4 −/− mice. Data are representative of one experiment reproduced four times ( n = 3–5), expressed as the mean ± SEM. Correlation between the frequency of CD4 + and CD4 + Foxp3 + cells from lungs of infected WT and CCR4 −/− mice h , i . Data are representative of four-independent experiments ( n = 22–25). All data are expressed as the mean ± SEM. Symbols represent individual animals and bars show the difference ( P
Figure Legend Snippet: CCR4 increases lung CD4 + Foxp3 + cells during M. tuberculosis infection. WT (white circles) and CCR4 −/− (black circles) mice were infected with M. tuberculosis as described in the Fig. 1 . At 15, 30 and 70 days of infection (dpi) the lungs were collected. Representative analysis of CD4 + and CD4 + Foxp3 + cells a . Frequency and total number of CD4 + cells b , c and CD4 + Foxp3 + cells d , e . Data are representative of three-independent experiments ( n = 16–22), expressed as the mean ± SEM. Ratio of frequency f and total number g of CD4 + and CD4 + Foxp3 + cells obtained from lungs of infected WT and CCR4 −/− mice. Data are representative of one experiment reproduced four times ( n = 3–5), expressed as the mean ± SEM. Correlation between the frequency of CD4 + and CD4 + Foxp3 + cells from lungs of infected WT and CCR4 −/− mice h , i . Data are representative of four-independent experiments ( n = 22–25). All data are expressed as the mean ± SEM. Symbols represent individual animals and bars show the difference ( P

Techniques Used: Infection, Mouse Assay

7) Product Images from "Murine DX5+NKT Cells Display Their Cytotoxic and Proapoptotic Potentials against Colitis-Inducing CD4+CD62Lhigh T Cells through Fas Ligand"

Article Title: Murine DX5+NKT Cells Display Their Cytotoxic and Proapoptotic Potentials against Colitis-Inducing CD4+CD62Lhigh T Cells through Fas Ligand

Journal: Journal of Immunology Research

doi: 10.1155/2018/8175810

Intracellular flow cytometry analysis of caspase-3 in CD4 + CD62L high cells after 10 h and 48 h of monoculture or coculture with CD8 + T or CD3 + DX5 + NKT cells. Results are given as mean + SEM. Experiments were repeated at least three times ( ∗ P
Figure Legend Snippet: Intracellular flow cytometry analysis of caspase-3 in CD4 + CD62L high cells after 10 h and 48 h of monoculture or coculture with CD8 + T or CD3 + DX5 + NKT cells. Results are given as mean + SEM. Experiments were repeated at least three times ( ∗ P

Techniques Used: Flow Cytometry, Cytometry

Flow cytometry analysis FasL expression of CD8 T and CD3 + DX5 + NKT cells after 4 h and 10 h monoculture or coculture with CD4 + CD62L high cells. Results are given as mean + SEM. Experiments were repeated at least three times ( ∗ P
Figure Legend Snippet: Flow cytometry analysis FasL expression of CD8 T and CD3 + DX5 + NKT cells after 4 h and 10 h monoculture or coculture with CD4 + CD62L high cells. Results are given as mean + SEM. Experiments were repeated at least three times ( ∗ P

Techniques Used: Flow Cytometry, Cytometry, Expressing

Intracellular flow cytometry analysis of caspase-3 in CD4 + CD62L high cells after 48 h of coculture with CD3 + DX5 + NKT cells and pretreatment with either FasL block or isotype control. Results are given as mean + SEM. Experiments were repeated at least three times ( ∗ P
Figure Legend Snippet: Intracellular flow cytometry analysis of caspase-3 in CD4 + CD62L high cells after 48 h of coculture with CD3 + DX5 + NKT cells and pretreatment with either FasL block or isotype control. Results are given as mean + SEM. Experiments were repeated at least three times ( ∗ P

Techniques Used: Flow Cytometry, Cytometry, Blocking Assay

Flow cytometry analysis of the spleen CD3 + DX5 + NKT, CD8 + T, CD4 + CD62L high , and CD4 + CD62L low cells of Balb/c mice after separation by MACS and FACS sorting (a). Proliferation (b) and proliferation index (c) of CFSE-labeled CD4 + CD62L high cells after 48 h and 96 h of monoculture or coculture with CD8 + T cells or CD3 + DX5 + NKT. Results are given as mean + SEM. Experiments were repeated at least three times ( ∗ P
Figure Legend Snippet: Flow cytometry analysis of the spleen CD3 + DX5 + NKT, CD8 + T, CD4 + CD62L high , and CD4 + CD62L low cells of Balb/c mice after separation by MACS and FACS sorting (a). Proliferation (b) and proliferation index (c) of CFSE-labeled CD4 + CD62L high cells after 48 h and 96 h of monoculture or coculture with CD8 + T cells or CD3 + DX5 + NKT. Results are given as mean + SEM. Experiments were repeated at least three times ( ∗ P

Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Magnetic Cell Separation, FACS, Labeling

Intracellular flow cytometry detection of IFN- γ in CD4 + CD62L high (a) and CD4 + CD62L low cells (b) after 4 h and 10 h of monoculture or coculture with CD8 + T cells or CD3 + DX5 + NKT. Results are given as mean + SEM. Experiments were repeated at least three times ( ∗ P
Figure Legend Snippet: Intracellular flow cytometry detection of IFN- γ in CD4 + CD62L high (a) and CD4 + CD62L low cells (b) after 4 h and 10 h of monoculture or coculture with CD8 + T cells or CD3 + DX5 + NKT. Results are given as mean + SEM. Experiments were repeated at least three times ( ∗ P

Techniques Used: Flow Cytometry, Cytometry

8) Product Images from "Actin stabilizer TAGLN2 potentiates adoptive T cell therapy by boosting the inside-out costimulation via lymphocyte function-associated antigen-1"

Article Title: Actin stabilizer TAGLN2 potentiates adoptive T cell therapy by boosting the inside-out costimulation via lymphocyte function-associated antigen-1

Journal: Oncoimmunology

doi: 10.1080/2162402X.2018.1500674

Overexpression of ICAM-1 in B16F10 cells restored the adhesion and cytokine release of TG2P-CD8 + T cells. (a) GFP + B16F10 and ICAM-1_GFP + B16F10 cells. The expression pattern of GFP or ICAM-1_GFP was determined in each cell line by flow cytometry and confocal microscopy. (b) Conjugate formation. OTI non-CD8 + T or OTI TG2P-CD8 + T cells (1 × 10 6 ) were incubated for 2 h with GFP + B16F10 or ICAM-1_GFP + B16F10 cells (1 × 10 6 ) in the presence of OVA peptides, and the percentages of conjugates were then determined by flow cytometry (left). The results are presented as bar graphs (right). In some cases, control IgG or anti-LFA-1 antibodies were used. Data are representative of at least three independent experiments. * P
Figure Legend Snippet: Overexpression of ICAM-1 in B16F10 cells restored the adhesion and cytokine release of TG2P-CD8 + T cells. (a) GFP + B16F10 and ICAM-1_GFP + B16F10 cells. The expression pattern of GFP or ICAM-1_GFP was determined in each cell line by flow cytometry and confocal microscopy. (b) Conjugate formation. OTI non-CD8 + T or OTI TG2P-CD8 + T cells (1 × 10 6 ) were incubated for 2 h with GFP + B16F10 or ICAM-1_GFP + B16F10 cells (1 × 10 6 ) in the presence of OVA peptides, and the percentages of conjugates were then determined by flow cytometry (left). The results are presented as bar graphs (right). In some cases, control IgG or anti-LFA-1 antibodies were used. Data are representative of at least three independent experiments. * P

Techniques Used: Over Expression, Expressing, Flow Cytometry, Cytometry, Confocal Microscopy, Incubation

Recombinant TAGLN2 fused with the protein transduction domain (TG2P) potentiated T-cell adhesion to APCs and cytokine release. (a) Schematic diagram of TG2P and amino acid (a.a.) sequences consisting of the C-terminus of TAGLN2, linker a.a. sequences, and N-terminus of PTD. (b, c) Transduction efficiency of TG2P, LPS-cleared TG2P (TG2P (-LPS)), and wild-type TG2 without PTD (TG2) in CD3 + T cells. CD3 + T cells were incubated with the indicated concentrations of each recombinant proteins for 4 h, and the cells were then subjected to western blot analysis. (d) The cells from (b) were cultured for the indicated times, and the retention time of TG2P in CD3 + T cells was analyzed. (e) Conjugate formation. TG2P-CD3 + T cells (1 × 10 6 ) were incubated with SEB-loaded B cells (1 × 10 6 ) for 30 min, and the percentages of conjugates were determined by flow cytometry (left). The results are presented as bar graphs. In some cases, control IgG or anti-LFA-1 antibodies were used. Data are representative of at least three independent experiments (b–e), * P
Figure Legend Snippet: Recombinant TAGLN2 fused with the protein transduction domain (TG2P) potentiated T-cell adhesion to APCs and cytokine release. (a) Schematic diagram of TG2P and amino acid (a.a.) sequences consisting of the C-terminus of TAGLN2, linker a.a. sequences, and N-terminus of PTD. (b, c) Transduction efficiency of TG2P, LPS-cleared TG2P (TG2P (-LPS)), and wild-type TG2 without PTD (TG2) in CD3 + T cells. CD3 + T cells were incubated with the indicated concentrations of each recombinant proteins for 4 h, and the cells were then subjected to western blot analysis. (d) The cells from (b) were cultured for the indicated times, and the retention time of TG2P in CD3 + T cells was analyzed. (e) Conjugate formation. TG2P-CD3 + T cells (1 × 10 6 ) were incubated with SEB-loaded B cells (1 × 10 6 ) for 30 min, and the percentages of conjugates were determined by flow cytometry (left). The results are presented as bar graphs. In some cases, control IgG or anti-LFA-1 antibodies were used. Data are representative of at least three independent experiments (b–e), * P

Techniques Used: Recombinant, Transduction, Incubation, Western Blot, Cell Culture, Flow Cytometry, Cytometry

TAGLN2 physically interacted with LFA-1 and increased Rap1 activity. (a) Localization of TAGLN2 (TG2), F-actin, and ICAM-1 (IC1) at the interface between T and B cells. Jurkat T cells expressing TG2_GFP and LifeA_mRFP (red) were conjugated with SEE-loaded Raji B cells stained with IC1_Cy5 (white) for 30 min. Three-dimensional reconstruction revealed the en face positions of contact interface areas between cells. Colocalization of TG2 and LifeA or TG2 and IC1 signals was determined by Pearson’s correlation coefficient (R). (b) Jurkat T cells expressing GFP and TG2_GFP were stimulated with anti-CD3/28 for 5 min. F-actin content was quantified using flow cytometry. Data are presented as relative fluorescence intensity compared with that in Jurkat T cells expressing GFP at 0 min. (c) Conjugate formation between Jurkat T cells expressing GFP or TG2_GFP cells and SEE-loaded Raji B cells. (d) Jurkat T cells were stimulated with anti-CD3/28 for the indicated times. Samples were immunoprecipitated with TS1/18 (anti-LFA-1 antibodies) and blotted with antibodies against the indicated proteins. (e) HEK293T cells were cotransfected with LFA-1 and different mutants of TG2, and immunoprecipitation and western blotting were performed. The schematic diagram shows the deletion mutants of TAGLN2 (M1, M2, and M3). (f) Activity of Rap1. Jurkat T cells expressing GFP and TG2_GFP were stimulated with anti-CD3/28 antibodies, and pull-down assays were performed. GTP-bound Rap1 was visualized by immunoblotting using anti-Rap1 antibodies. Data are representative of three independent experiments (b–f) (g) TG2 expression in CD4 + or CD8 + T cells from normal or severe tumor-bearing mice. When tumor size of the mice was over 3,000 mm 3 , the mice were defined as “severe tumor-bearing mice”. CD4 + or CD8 + T cells were purified from spleen and lymph nodes of mice and then subjected to western blot analysis. Intensities of western blot bands were analyzed by the Quantity one image analysis program (Bio-Rad) and were normalized to β-actin. * P
Figure Legend Snippet: TAGLN2 physically interacted with LFA-1 and increased Rap1 activity. (a) Localization of TAGLN2 (TG2), F-actin, and ICAM-1 (IC1) at the interface between T and B cells. Jurkat T cells expressing TG2_GFP and LifeA_mRFP (red) were conjugated with SEE-loaded Raji B cells stained with IC1_Cy5 (white) for 30 min. Three-dimensional reconstruction revealed the en face positions of contact interface areas between cells. Colocalization of TG2 and LifeA or TG2 and IC1 signals was determined by Pearson’s correlation coefficient (R). (b) Jurkat T cells expressing GFP and TG2_GFP were stimulated with anti-CD3/28 for 5 min. F-actin content was quantified using flow cytometry. Data are presented as relative fluorescence intensity compared with that in Jurkat T cells expressing GFP at 0 min. (c) Conjugate formation between Jurkat T cells expressing GFP or TG2_GFP cells and SEE-loaded Raji B cells. (d) Jurkat T cells were stimulated with anti-CD3/28 for the indicated times. Samples were immunoprecipitated with TS1/18 (anti-LFA-1 antibodies) and blotted with antibodies against the indicated proteins. (e) HEK293T cells were cotransfected with LFA-1 and different mutants of TG2, and immunoprecipitation and western blotting were performed. The schematic diagram shows the deletion mutants of TAGLN2 (M1, M2, and M3). (f) Activity of Rap1. Jurkat T cells expressing GFP and TG2_GFP were stimulated with anti-CD3/28 antibodies, and pull-down assays were performed. GTP-bound Rap1 was visualized by immunoblotting using anti-Rap1 antibodies. Data are representative of three independent experiments (b–f) (g) TG2 expression in CD4 + or CD8 + T cells from normal or severe tumor-bearing mice. When tumor size of the mice was over 3,000 mm 3 , the mice were defined as “severe tumor-bearing mice”. CD4 + or CD8 + T cells were purified from spleen and lymph nodes of mice and then subjected to western blot analysis. Intensities of western blot bands were analyzed by the Quantity one image analysis program (Bio-Rad) and were normalized to β-actin. * P

Techniques Used: Activity Assay, Expressing, Staining, Flow Cytometry, Cytometry, Fluorescence, Immunoprecipitation, Western Blot, Mouse Assay, Purification

Transduction of TG2P in CD8 + T cells increased adhesion and cytokine release in response to ICAM-1-positive cancer cells. (a) Conjugate formation. OTI non-CD8 + T or OTI TG2P-CD8 + T cells (1 × 10 6 ) were incubated for 2 h with E0771 cells (1 × 10 6 ) in the absence or presence of OVA peptides, and the percentages of conjugates were then determined by flow cytometry (left). The results are presented as bar graphs (right). In some cases, control IgG or anti-LFA-1 antibodies were used. Data are representative of at least three independent experiments. * P
Figure Legend Snippet: Transduction of TG2P in CD8 + T cells increased adhesion and cytokine release in response to ICAM-1-positive cancer cells. (a) Conjugate formation. OTI non-CD8 + T or OTI TG2P-CD8 + T cells (1 × 10 6 ) were incubated for 2 h with E0771 cells (1 × 10 6 ) in the absence or presence of OVA peptides, and the percentages of conjugates were then determined by flow cytometry (left). The results are presented as bar graphs (right). In some cases, control IgG or anti-LFA-1 antibodies were used. Data are representative of at least three independent experiments. * P

Techniques Used: Transduction, Incubation, Flow Cytometry, Cytometry

9) Product Images from "Distinct Role of IL-27 in Immature and LPS-Induced Mature Dendritic Cell-Mediated Development of CD4+ CD127+3G11+ Regulatory T Cell Subset"

Article Title: Distinct Role of IL-27 in Immature and LPS-Induced Mature Dendritic Cell-Mediated Development of CD4+ CD127+3G11+ Regulatory T Cell Subset

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02562

IL-27 facilitates LPS-stimulated mature DC-mediated development of CD4 + CD127 + 3G11 + T reg subset ex vivo . Bone marrow-derived DCs were pulsed with MOG peptide and treated with IL-27 (20 ng/ml, 72 h) (DCs+IL-27) or LPS (DCs+LPS) (1 μg/ml, 24 h) or both LPS and IL-27 (DCs+LPS+IL-27). These DCs were then i.v. transferred into mice with EAE shown in Figure 4 . Mice treated with PBS are control. Spleen cells were isolated and re-stimulated with MOG peptide (0.1 μM) and IL-2 (1 ng/ml) for 72 h. Cells were collected and CD4 + CD25 + FoxP3 + GITR + T regs were gated. The frequency of CD127 + 3G11 + cells is demonstrated. T lymphocytes incubated with isotype control antibodies are isotype control. Error bars indicated in this figure represent mean and SD of frequency of CD4 + CD127 + 3G11 + cells in three independent experiments [ n = 3, t test, P (DC, DC+IL−27) = 0.8682; P (DC+LPS, DC+LPS+IL−27) = 0.0105].
Figure Legend Snippet: IL-27 facilitates LPS-stimulated mature DC-mediated development of CD4 + CD127 + 3G11 + T reg subset ex vivo . Bone marrow-derived DCs were pulsed with MOG peptide and treated with IL-27 (20 ng/ml, 72 h) (DCs+IL-27) or LPS (DCs+LPS) (1 μg/ml, 24 h) or both LPS and IL-27 (DCs+LPS+IL-27). These DCs were then i.v. transferred into mice with EAE shown in Figure 4 . Mice treated with PBS are control. Spleen cells were isolated and re-stimulated with MOG peptide (0.1 μM) and IL-2 (1 ng/ml) for 72 h. Cells were collected and CD4 + CD25 + FoxP3 + GITR + T regs were gated. The frequency of CD127 + 3G11 + cells is demonstrated. T lymphocytes incubated with isotype control antibodies are isotype control. Error bars indicated in this figure represent mean and SD of frequency of CD4 + CD127 + 3G11 + cells in three independent experiments [ n = 3, t test, P (DC, DC+IL−27) = 0.8682; P (DC+LPS, DC+LPS+IL−27) = 0.0105].

Techniques Used: Ex Vivo, Derivative Assay, Mouse Assay, Isolation, Incubation

i.v. transfer of IL-27-treated mature DCs induced by LPS inhibits immune tolerance mediated by LPS-stimulated DCs in vivo . C57 BL/6J mice were immunized with MOG peptide (200 μg/per mouse) and CFA. Immature dendritic cells (DCs) were incubated with IL-27 (DCs+IL-27) or LPS (DCs+LPS) or both IL-27 and LPS (DCs+LPS+IL-27). Mice in control group were i.v. transferred with PBS. EAE was then induced and shown by clinical score. Error bars in this figure represent mean and SEM of triplicate determinations of EAE clinical score in one experiment ( n = 3, two-way ANOVA test, P (DC, DC+IL−27) = 0.7960; P (DC+LPS, DC+LPS+IL−27) = 0.0001; NS, no significant difference).
Figure Legend Snippet: i.v. transfer of IL-27-treated mature DCs induced by LPS inhibits immune tolerance mediated by LPS-stimulated DCs in vivo . C57 BL/6J mice were immunized with MOG peptide (200 μg/per mouse) and CFA. Immature dendritic cells (DCs) were incubated with IL-27 (DCs+IL-27) or LPS (DCs+LPS) or both IL-27 and LPS (DCs+LPS+IL-27). Mice in control group were i.v. transferred with PBS. EAE was then induced and shown by clinical score. Error bars in this figure represent mean and SEM of triplicate determinations of EAE clinical score in one experiment ( n = 3, two-way ANOVA test, P (DC, DC+IL−27) = 0.7960; P (DC+LPS, DC+LPS+IL−27) = 0.0001; NS, no significant difference).

Techniques Used: In Vivo, Mouse Assay, Incubation

10) Product Images from "Early heme oxygenase 1 induction delays tumour initiation and enhances DNA damage repair in liver macrophages of Mdr2−/− mice"

Article Title: Early heme oxygenase 1 induction delays tumour initiation and enhances DNA damage repair in liver macrophages of Mdr2−/− mice

Journal: Scientific Reports

doi: 10.1038/s41598-018-33233-0

HO-1 induction reduces DNA damage in macrophages in vitro and in vivo . Representative images (20x) of tissue sections of 65-week-old Mdr2 −/− mice, treated as described in Suppl. Fig. 1A and WT mice of the same age stained for ( A ) DAPI, γH2AX, and the macrophage marker F4/80 as well as ( B ) DAPI, γH2AX, and the hepatocyte marker HNF4-alpha. ( C ) Quantification of γH2AX + F4/80 + macrophages and γH2AX + HNF4-alpha + hepatocytes in tissue sections described in A (n ≥ 4 HPF/slide). ( D ) Ho-1 mRNA expression levels of BMDMs derived from 14-week-old WT and Mdr2 −/− mice, with or without irradiation, with or without CoPP treatment [10 µg/ml; 24 h prior to irradiation] of one representative experiment (n = 3). ( E ) Frequency distribution of γH2AX + foci in BMDMs (65w; WT, Mdr2 −/− ) with or without CoPP treatment in vitro [10 µg/ml; 24 h]. ( F ) Hepatic mRNA expression levels of Cd36 determined by quantitative real time RT-PCR in livers of mice described in Suppl. Fig. 1A . ( G ) Quantification of phagocytic activity of BMDMs derived from 14-week-old animals described in Suppl. Fig. 1A determined by flow cytometry (n ≥ 3; one representative experiment). Data expressed as means ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.
Figure Legend Snippet: HO-1 induction reduces DNA damage in macrophages in vitro and in vivo . Representative images (20x) of tissue sections of 65-week-old Mdr2 −/− mice, treated as described in Suppl. Fig. 1A and WT mice of the same age stained for ( A ) DAPI, γH2AX, and the macrophage marker F4/80 as well as ( B ) DAPI, γH2AX, and the hepatocyte marker HNF4-alpha. ( C ) Quantification of γH2AX + F4/80 + macrophages and γH2AX + HNF4-alpha + hepatocytes in tissue sections described in A (n ≥ 4 HPF/slide). ( D ) Ho-1 mRNA expression levels of BMDMs derived from 14-week-old WT and Mdr2 −/− mice, with or without irradiation, with or without CoPP treatment [10 µg/ml; 24 h prior to irradiation] of one representative experiment (n = 3). ( E ) Frequency distribution of γH2AX + foci in BMDMs (65w; WT, Mdr2 −/− ) with or without CoPP treatment in vitro [10 µg/ml; 24 h]. ( F ) Hepatic mRNA expression levels of Cd36 determined by quantitative real time RT-PCR in livers of mice described in Suppl. Fig. 1A . ( G ) Quantification of phagocytic activity of BMDMs derived from 14-week-old animals described in Suppl. Fig. 1A determined by flow cytometry (n ≥ 3; one representative experiment). Data expressed as means ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Techniques Used: In Vitro, In Vivo, Mouse Assay, Staining, Marker, Expressing, Derivative Assay, Irradiation, Quantitative RT-PCR, Activity Assay, Flow Cytometry, Cytometry

11) Product Images from "HIF-1α Is a Metabolic Switch between Glycolytic-Driven Migration and Oxidative Phosphorylation-Driven Immunosuppression of Tregs in Glioblastoma"

Article Title: HIF-1α Is a Metabolic Switch between Glycolytic-Driven Migration and Oxidative Phosphorylation-Driven Immunosuppression of Tregs in Glioblastoma

Journal: Cell reports

doi: 10.1016/j.celrep.2019.03.029

Regulatory T Cells Utilize Lipids for Their Mitochondrial Metabolism under Hypoxia and Prefer Lipids for Metabolism within Glioma (A and B) Flow-cytometry-sorted and expanded HIF-1α WT or HIF-1α KO Tregs were placed under 1% O 2 overnight before being adhered to microplates, and extracellular flux analysis was performed over time (A) and analyzed as bar graphs (B). Eto treatment (20 μm) was added immediately before assay to determine reliance on lipids for mitochondrial metabolism. (C–F) Wild-type C57/Bl6 mice were implanted with 4 × 10 5 GL-261 astrocytoma cells, and after 2 weeks of tumor growth, T cell expression of surface fatty acid transporters was analyzed via flow cytometry. (C) Percent palmitic acid uptake and MFI of conventional CD4 + , CD8 + , and Treg subsets. (D) Percent 2-NDBG and MFI of 2-NBDG conventional CD4 + , CD8 + , and Treg subsets. (E) Tumor interstitial fluid was obtained from either the tumor hemisphere or the non-tumor hemisphere of mice, and FFA content was measured via colorimetric readout. (F) Data show the expression of fatty acid transporters CD36, SLC27A1, and SLC27A4 across different T cell subsets in the brains, DLN, and spleens of tumor-bearing mice (left); representative flow cytometry plots are shown on the right. Statistics were calculated as percent positive population ± SEM. Data in (A) and (B) were analyzed using Wave software from Agilent. Statistics were calculated as percent positive population ± SEM, n = 5 per group; results are representative of three experiments in (A) and (B) and two experiments in (C)–(F). One-way ANOVA followed by Tukey’s post hoc analysis was used to calculate significance. *p
Figure Legend Snippet: Regulatory T Cells Utilize Lipids for Their Mitochondrial Metabolism under Hypoxia and Prefer Lipids for Metabolism within Glioma (A and B) Flow-cytometry-sorted and expanded HIF-1α WT or HIF-1α KO Tregs were placed under 1% O 2 overnight before being adhered to microplates, and extracellular flux analysis was performed over time (A) and analyzed as bar graphs (B). Eto treatment (20 μm) was added immediately before assay to determine reliance on lipids for mitochondrial metabolism. (C–F) Wild-type C57/Bl6 mice were implanted with 4 × 10 5 GL-261 astrocytoma cells, and after 2 weeks of tumor growth, T cell expression of surface fatty acid transporters was analyzed via flow cytometry. (C) Percent palmitic acid uptake and MFI of conventional CD4 + , CD8 + , and Treg subsets. (D) Percent 2-NDBG and MFI of 2-NBDG conventional CD4 + , CD8 + , and Treg subsets. (E) Tumor interstitial fluid was obtained from either the tumor hemisphere or the non-tumor hemisphere of mice, and FFA content was measured via colorimetric readout. (F) Data show the expression of fatty acid transporters CD36, SLC27A1, and SLC27A4 across different T cell subsets in the brains, DLN, and spleens of tumor-bearing mice (left); representative flow cytometry plots are shown on the right. Statistics were calculated as percent positive population ± SEM. Data in (A) and (B) were analyzed using Wave software from Agilent. Statistics were calculated as percent positive population ± SEM, n = 5 per group; results are representative of three experiments in (A) and (B) and two experiments in (C)–(F). One-way ANOVA followed by Tukey’s post hoc analysis was used to calculate significance. *p

Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Expressing, Software

In Vivo Treatment of Eto Causes a Survival Benefit in Immunocompetent Mice (A) Survival of WT or immunodeficient Rag 0/0 mice implanted with 4 × 10 5 GL-261 and treated with intracranial Eto administration beginning at day 7 after tumor implantation. (B) Quantification of Tregs and their ratios to other T cell subsets 48 hr after Eto treatment (day 9). (C) Survival of mice injected i.p. with 30 mg/kg Eto. (D) Flow-cytometric analysis of Treg infiltration and their ratios to other T cell subsets 48 h after second Eto treatment (9 days). Survival curves are from at least 7 mice per group from two independent experiments; statistical significance was calculated using log-rank analysis. Flow cytometry statistics were calculated and are indicated as percent positive population ± SEM; n = 5 per group, representative of two independent experiments in (B) and one experiment in (D). Unpaired t test analysis was used to calculate significance. *p
Figure Legend Snippet: In Vivo Treatment of Eto Causes a Survival Benefit in Immunocompetent Mice (A) Survival of WT or immunodeficient Rag 0/0 mice implanted with 4 × 10 5 GL-261 and treated with intracranial Eto administration beginning at day 7 after tumor implantation. (B) Quantification of Tregs and their ratios to other T cell subsets 48 hr after Eto treatment (day 9). (C) Survival of mice injected i.p. with 30 mg/kg Eto. (D) Flow-cytometric analysis of Treg infiltration and their ratios to other T cell subsets 48 h after second Eto treatment (9 days). Survival curves are from at least 7 mice per group from two independent experiments; statistical significance was calculated using log-rank analysis. Flow cytometry statistics were calculated and are indicated as percent positive population ± SEM; n = 5 per group, representative of two independent experiments in (B) and one experiment in (D). Unpaired t test analysis was used to calculate significance. *p

Techniques Used: In Vivo, Mouse Assay, Tumor Implantation, Injection, Flow Cytometry, Cytometry

Inhibition of Either Lipid Uptake or Lipid Oxidation Prevents Immunosuppressive Capabilities of Regulatory T Cells (A) Sorted and expanded Tregs were cultured for 72 h in the presence of the fatty acid oxidation inhibitor etomoxir (200 μM) or the fatty acid uptake inhibitor SSO (200 μM), and the expression of various Tregs markers was assessed via flow cytometry. (B and E) Sorted Tregs were cultured for 72 h in the presence of etomoxir (Eto; 200 μM; B) or SSO (200 μM; E), and Foxp3 retention was determined. (C and D) Percent CD8 T cell proliferation with co-culture of Eto pre-treated Tregs shown as a histogram (C) and analyzed as bar graphs with reducing Treg ratios (D) that were enumerated via flow cytometry. (F and G) Percent CD8 T cell proliferation with co-culture of SSO pre-treated Tregs shown as a histogram (F) and analyzed as bar graphs with reducing Treg ratios (G) that were enumerated via flow cytometry. (H and I) Sorted and expanded WT or HIF-1α KO Tregs were pretreated with (H) Eto (200 μM) or (I) SSO (200 μM) for 24 h before a suppressor assay was run under 1% O 2 . After 72 h, expansion indexes were determined via flow cytometry. Flow cytometry statistics were calculated and shown as percent positive or MFI ± SEM; n = 5 per group in (A), and n = 3 per ratio in (B)–(I), representative of 2–3 independent experiments. A one-way ANOVA followed by Tukey’s post hoc analysis was used to calculate significance. *p
Figure Legend Snippet: Inhibition of Either Lipid Uptake or Lipid Oxidation Prevents Immunosuppressive Capabilities of Regulatory T Cells (A) Sorted and expanded Tregs were cultured for 72 h in the presence of the fatty acid oxidation inhibitor etomoxir (200 μM) or the fatty acid uptake inhibitor SSO (200 μM), and the expression of various Tregs markers was assessed via flow cytometry. (B and E) Sorted Tregs were cultured for 72 h in the presence of etomoxir (Eto; 200 μM; B) or SSO (200 μM; E), and Foxp3 retention was determined. (C and D) Percent CD8 T cell proliferation with co-culture of Eto pre-treated Tregs shown as a histogram (C) and analyzed as bar graphs with reducing Treg ratios (D) that were enumerated via flow cytometry. (F and G) Percent CD8 T cell proliferation with co-culture of SSO pre-treated Tregs shown as a histogram (F) and analyzed as bar graphs with reducing Treg ratios (G) that were enumerated via flow cytometry. (H and I) Sorted and expanded WT or HIF-1α KO Tregs were pretreated with (H) Eto (200 μM) or (I) SSO (200 μM) for 24 h before a suppressor assay was run under 1% O 2 . After 72 h, expansion indexes were determined via flow cytometry. Flow cytometry statistics were calculated and shown as percent positive or MFI ± SEM; n = 5 per group in (A), and n = 3 per ratio in (B)–(I), representative of 2–3 independent experiments. A one-way ANOVA followed by Tukey’s post hoc analysis was used to calculate significance. *p

Techniques Used: Inhibition, Cell Culture, Expressing, Flow Cytometry, Cytometry, Co-Culture Assay

HIF-1α KO Tregs Suppress CD8 + T Cell Proliferation Better than HIF-1α WT Tregs under Hypoxia due to Enhanced Glucose Oxidation (A and B) Sorted and expanded Tregs were plated with proliferation-dye-labeled CD8 + T cells at decreasing ratios to determine their suppressive capability under (A) 21% O 2 and (B) 1% O 2 . After 72 h, CD8 + T cell proliferation was analyzed. An n of 3 wells per ratio was analyzed, representative of three independent experiments. (C) Sorted and expanded Tregs were pre-treated with UK5099 (10 μm) or vehicle control for 24 h before a suppressor assay was run under 1% O 2 . After 72 h, percent proliferation and expansion indexes were determined via flow cytometry. An n of 3 per condition was analyzed, from two independent experiments. (D) Sorted and expanded Tregs were pretreated with 1 mM DCA treatment overnight before suppressor assays were performed under 1% O 2 . Statistics were calculated as percent positive population ± SEM. Unpaired t test analysis was used to calculate significance. *p
Figure Legend Snippet: HIF-1α KO Tregs Suppress CD8 + T Cell Proliferation Better than HIF-1α WT Tregs under Hypoxia due to Enhanced Glucose Oxidation (A and B) Sorted and expanded Tregs were plated with proliferation-dye-labeled CD8 + T cells at decreasing ratios to determine their suppressive capability under (A) 21% O 2 and (B) 1% O 2 . After 72 h, CD8 + T cell proliferation was analyzed. An n of 3 wells per ratio was analyzed, representative of three independent experiments. (C) Sorted and expanded Tregs were pre-treated with UK5099 (10 μm) or vehicle control for 24 h before a suppressor assay was run under 1% O 2 . After 72 h, percent proliferation and expansion indexes were determined via flow cytometry. An n of 3 per condition was analyzed, from two independent experiments. (D) Sorted and expanded Tregs were pretreated with 1 mM DCA treatment overnight before suppressor assays were performed under 1% O 2 . Statistics were calculated as percent positive population ± SEM. Unpaired t test analysis was used to calculate significance. *p

Techniques Used: Labeling, Flow Cytometry, Cytometry

Conditional Knockout of HIF-1α in Foxp3 + T Cells Inhibits Migration of Tregs to Brain Tumors In Vivo (A) To test Treg migration in vivo , splenic-sorted and expanded Tregs were co-labeled with eFluor 450 (HIF-1α WT) and eFluor 670 (HIF-1α KO) cell proliferation dyes and injected i.v. at a 1:1 ratio into mice harboring GL-261. (B) After 48 h, the brain, spleen, and DLN of mice injected with Tregs were isolated, and the ratio of control to HIF-1α KO Tregs was determined. (C) HIF-1α WT or HIF-1α KO mice were implanted with 4 × 10 5 GL-261 astrocytoma cells, and overall survival was determined. (D) After 2 weeks of tumor growth, Treg abundance was analyzed via flow cytometry. (E) Abundance of Foxp3 + cells from tumor-bearing mice was quantified in tissue sections. Kaplan-Meier curves are n = 7 per group from two independent experiments, and significance was calculated using log-rank analysis. Flow cytometry statistics shown as percent positive population ± SEM; n = 5 per group, representative of two experiments. In (B), the ratios of WT/HIF-1α KO Tregs were indicated as mean ± SEM. In (E), 3–5 fields per section were quantified for Foxp3 + DAPI + nuclei. n = 3 mice per group. Unpaired t test analysis was used to calculate significance. *p
Figure Legend Snippet: Conditional Knockout of HIF-1α in Foxp3 + T Cells Inhibits Migration of Tregs to Brain Tumors In Vivo (A) To test Treg migration in vivo , splenic-sorted and expanded Tregs were co-labeled with eFluor 450 (HIF-1α WT) and eFluor 670 (HIF-1α KO) cell proliferation dyes and injected i.v. at a 1:1 ratio into mice harboring GL-261. (B) After 48 h, the brain, spleen, and DLN of mice injected with Tregs were isolated, and the ratio of control to HIF-1α KO Tregs was determined. (C) HIF-1α WT or HIF-1α KO mice were implanted with 4 × 10 5 GL-261 astrocytoma cells, and overall survival was determined. (D) After 2 weeks of tumor growth, Treg abundance was analyzed via flow cytometry. (E) Abundance of Foxp3 + cells from tumor-bearing mice was quantified in tissue sections. Kaplan-Meier curves are n = 7 per group from two independent experiments, and significance was calculated using log-rank analysis. Flow cytometry statistics shown as percent positive population ± SEM; n = 5 per group, representative of two experiments. In (B), the ratios of WT/HIF-1α KO Tregs were indicated as mean ± SEM. In (E), 3–5 fields per section were quantified for Foxp3 + DAPI + nuclei. n = 3 mice per group. Unpaired t test analysis was used to calculate significance. *p

Techniques Used: Knock-Out, Migration, In Vivo, Labeling, Injection, Mouse Assay, Isolation, Flow Cytometry, Cytometry

12) Product Images from "Surfaceome interrogation using an RNA-seq approach highlights leukemia initiating cell biomarkers in an LMO2 T cell transgenic model"

Article Title: Surfaceome interrogation using an RNA-seq approach highlights leukemia initiating cell biomarkers in an LMO2 T cell transgenic model

Journal: Scientific Reports

doi: 10.1038/s41598-019-42214-w

Flow cytometry analysis of CD53 and CD59a expression on mouse Lck-Lmo2 DN thymocytes. Thymus cells were prepared from asymptomatic Lck-Lmo2 mice or equivalent age wild type mice and expression of CD53 (panel A; wild type mice 15 weeks old; Lck-Lmo2 21 weeks old) or CD59a (panel B; wild type 16 weeks old, Lck-Lmo2 19 weeks old) was profiled in the CD4-/CD8- DN subsets (The histogram shows the results for DN cells on stained cells (black) and cells stained with isotype control (light grey). The following fluorescent antibodies were used: CD90-V450, CD4-PE-cy7, CD8-PE, CD44-V500, CD25-APC, CD53-BB515. The x-axis shows fluorescence on a log scale. Panels C,D show the flow cytometry data for the same mice comparing the expression of CD53 (panel C) and CD59a (panel D) in single CD4 positive (SP), single CD8 positive and CD4/CD8 double positive (DP) thymocytes. NB: No suitable antibody is available binding to mouse GPR56.
Figure Legend Snippet: Flow cytometry analysis of CD53 and CD59a expression on mouse Lck-Lmo2 DN thymocytes. Thymus cells were prepared from asymptomatic Lck-Lmo2 mice or equivalent age wild type mice and expression of CD53 (panel A; wild type mice 15 weeks old; Lck-Lmo2 21 weeks old) or CD59a (panel B; wild type 16 weeks old, Lck-Lmo2 19 weeks old) was profiled in the CD4-/CD8- DN subsets (The histogram shows the results for DN cells on stained cells (black) and cells stained with isotype control (light grey). The following fluorescent antibodies were used: CD90-V450, CD4-PE-cy7, CD8-PE, CD44-V500, CD25-APC, CD53-BB515. The x-axis shows fluorescence on a log scale. Panels C,D show the flow cytometry data for the same mice comparing the expression of CD53 (panel C) and CD59a (panel D) in single CD4 positive (SP), single CD8 positive and CD4/CD8 double positive (DP) thymocytes. NB: No suitable antibody is available binding to mouse GPR56.

Techniques Used: Flow Cytometry, Cytometry, Expressing, Mouse Assay, Staining, Fluorescence, Binding Assay

13) Product Images from "cGAS/STING/TBK1/IRF3 Signaling Pathway Activates BMDCs Maturation Following Mycobacterium bovis Infection"

Article Title: cGAS/STING/TBK1/IRF3 Signaling Pathway Activates BMDCs Maturation Following Mycobacterium bovis Infection

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20040895

Mediation of the maturation and activation of BMDCs during Mycobacterium bovis infection by the cGAS pathway. Mycobacterium bovis regulated the maturation and activation of BMDCs via the cGAS/STING/TBK1/IRF3-dependent pathway and promoted type I IFN production. Furthermore, type I IFN and its receptor IFNAR contributed to this process. Moreover, the mature and activated BMDCs enhanced the proliferation of T cells by some surface markers and cytokines. These signaling pathways linked the innate and adaptive immune responses. (The full line arrows signify promotion; the dotted arrows signify releasing cytokines.)
Figure Legend Snippet: Mediation of the maturation and activation of BMDCs during Mycobacterium bovis infection by the cGAS pathway. Mycobacterium bovis regulated the maturation and activation of BMDCs via the cGAS/STING/TBK1/IRF3-dependent pathway and promoted type I IFN production. Furthermore, type I IFN and its receptor IFNAR contributed to this process. Moreover, the mature and activated BMDCs enhanced the proliferation of T cells by some surface markers and cytokines. These signaling pathways linked the innate and adaptive immune responses. (The full line arrows signify promotion; the dotted arrows signify releasing cytokines.)

Techniques Used: Activation Assay, Infection

The cGAS pathway promotes the maturation and activation of BMDCs. ( A ) The cell surface markers of CD40, CD80, CD86, and MHC class II were analyzed by flow cytometry in BMDCs transfected with siCon or sicGAS and then infected for 24 h with M. bovis (MOI 5). The CD11c marker was used to set the gate for flow cytometric analysis. ( B ) The positive cell rate of surface markers in each group was calculated, and histograms were generated by FlowJo software. ( C ) Culture supernatants were collected after 24 h; the expressions of TNF-α, IL-6, IL-10, and IL-12p70 were assayed by ELISA. All data are expressed as mean ± SD, (* p
Figure Legend Snippet: The cGAS pathway promotes the maturation and activation of BMDCs. ( A ) The cell surface markers of CD40, CD80, CD86, and MHC class II were analyzed by flow cytometry in BMDCs transfected with siCon or sicGAS and then infected for 24 h with M. bovis (MOI 5). The CD11c marker was used to set the gate for flow cytometric analysis. ( B ) The positive cell rate of surface markers in each group was calculated, and histograms were generated by FlowJo software. ( C ) Culture supernatants were collected after 24 h; the expressions of TNF-α, IL-6, IL-10, and IL-12p70 were assayed by ELISA. All data are expressed as mean ± SD, (* p

Techniques Used: Activation Assay, Flow Cytometry, Cytometry, Transfection, Infection, Marker, Generated, Software, Enzyme-linked Immunosorbent Assay

The cyclic GMP-AMP synthase (cGAS) pathway is activated in bone marrow-derived dendritic cells (BMDCs) during Mycobacterium bovis infection. ( A ) BMDCs were treated with siRNA, siCon, and M. bovis , and cGAS protein was analyzed by Western blotting at 24 h after treatment. ( B ) The related proteins in the cGAS pathway in BMDCs were assayed by Western blotting. The protein levels of cGAS, p-STING, STING, TBK1, and p-TBK1 were analyzed in BMDCs transfected with siCon or sicGAS and then infected for 24 or 48 h with M. bovis (MOI 5). ( C ) The co-localization of IRF3 within the nucleus was detected by immunofluorescence microscopy (400 ×). ( D ) The culture supernatants were harvested after 24 h and assessed by ELISA. All data are expressed as mean ± SD, (* p
Figure Legend Snippet: The cyclic GMP-AMP synthase (cGAS) pathway is activated in bone marrow-derived dendritic cells (BMDCs) during Mycobacterium bovis infection. ( A ) BMDCs were treated with siRNA, siCon, and M. bovis , and cGAS protein was analyzed by Western blotting at 24 h after treatment. ( B ) The related proteins in the cGAS pathway in BMDCs were assayed by Western blotting. The protein levels of cGAS, p-STING, STING, TBK1, and p-TBK1 were analyzed in BMDCs transfected with siCon or sicGAS and then infected for 24 or 48 h with M. bovis (MOI 5). ( C ) The co-localization of IRF3 within the nucleus was detected by immunofluorescence microscopy (400 ×). ( D ) The culture supernatants were harvested after 24 h and assessed by ELISA. All data are expressed as mean ± SD, (* p

Techniques Used: Derivative Assay, Infection, Western Blot, Transfection, Immunofluorescence, Microscopy, Enzyme-linked Immunosorbent Assay

BMDCs promote T cell activity in the presence of type I interferons. ( A ) The proliferation of CD4 + T cells was analyzed by flow cytometry after 72 h co-culture. The interferon receptor was treated with neutralizing anti-mouse IFNAR mAb (10 ng/mL) to block and then infected for 24 h with M. bovis (MOI 5). CFSE-stained CD4 + T cells were co-cultured with three different BMDC groups, and the ratios of BMDC:T cells were 1:10 for 72 h. ( B ) After 72 h co-culture, the culture supernatants were harvested and assessed by ELISA. All data are expressed as mean ± SD. The number above the horizontal bar represents the proliferation rate of CD4 + T cell, (* p
Figure Legend Snippet: BMDCs promote T cell activity in the presence of type I interferons. ( A ) The proliferation of CD4 + T cells was analyzed by flow cytometry after 72 h co-culture. The interferon receptor was treated with neutralizing anti-mouse IFNAR mAb (10 ng/mL) to block and then infected for 24 h with M. bovis (MOI 5). CFSE-stained CD4 + T cells were co-cultured with three different BMDC groups, and the ratios of BMDC:T cells were 1:10 for 72 h. ( B ) After 72 h co-culture, the culture supernatants were harvested and assessed by ELISA. All data are expressed as mean ± SD. The number above the horizontal bar represents the proliferation rate of CD4 + T cell, (* p

Techniques Used: Activity Assay, Flow Cytometry, Cytometry, Co-Culture Assay, Blocking Assay, Infection, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

Type I interferon (IFN) and its receptor (IFNAR) contribute to the cGAS pathway in BMDCs. ( A ) The expression of IFN-β was assayed after anti-mouse IFNAR monoclonal antibody (mAb; 10 ng/mL) treatment. ( B ) The cell surface markers of CD40, CD80, CD86, and MHC class II were analyzed by flow cytometry in BMDCs transfected with siCon or sicGAS and then infected for 24 h with M. bovis (MOI 5). The CD11c marker was used to set the gate for flow cytometric analysis. The interferon receptor was treated with neutralizing anti-mouse IFNAR mAb (10 ng/mL) to block and then infected for 24 h with M. bovis (MOI 5). ( C ) The positive cell rate of surface markers in each group was calculated, and histograms were generated by FlowJo software. ( D ) Culture supernatants were harvested after 24 h; the expression of TNF-α, IL-6, IL-10, and IL-12p70 was assayed by ELISA. IFN-β + M. bovis : BMDCs were treated with exogenous IFN-β (10 ng/mL) and then infected with M. bovis [ 16 ]. All data are expressed as mean ± SD, (* p
Figure Legend Snippet: Type I interferon (IFN) and its receptor (IFNAR) contribute to the cGAS pathway in BMDCs. ( A ) The expression of IFN-β was assayed after anti-mouse IFNAR monoclonal antibody (mAb; 10 ng/mL) treatment. ( B ) The cell surface markers of CD40, CD80, CD86, and MHC class II were analyzed by flow cytometry in BMDCs transfected with siCon or sicGAS and then infected for 24 h with M. bovis (MOI 5). The CD11c marker was used to set the gate for flow cytometric analysis. The interferon receptor was treated with neutralizing anti-mouse IFNAR mAb (10 ng/mL) to block and then infected for 24 h with M. bovis (MOI 5). ( C ) The positive cell rate of surface markers in each group was calculated, and histograms were generated by FlowJo software. ( D ) Culture supernatants were harvested after 24 h; the expression of TNF-α, IL-6, IL-10, and IL-12p70 was assayed by ELISA. IFN-β + M. bovis : BMDCs were treated with exogenous IFN-β (10 ng/mL) and then infected with M. bovis [ 16 ]. All data are expressed as mean ± SD, (* p

Techniques Used: Expressing, Flow Cytometry, Cytometry, Transfection, Infection, Marker, Blocking Assay, Generated, Software, Enzyme-linked Immunosorbent Assay

14) Product Images from "Blockade of CTLA-4 and Tim-3 pathways induces fetal loss with altered cytokine profiles by decidual CD4+T cells"

Article Title: Blockade of CTLA-4 and Tim-3 pathways induces fetal loss with altered cytokine profiles by decidual CD4+T cells

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1251-0

Cytokine production in dCD4 + T cells during normal pregnancy. a Production of the pro-inflammatory cytokines TNF-α, IFN-γ and IL-17A in dCTLA-4 + Tim-3 + CD4 + T cells and dCTLA-4 − Tim-3 − CD4 + T cells from the first trimester of human normal pregnancies. n = 9. b Quantitation of flow cytometric analysis of IL-4, TGF-β1, and IL-10 of dCTLA-4 + Tim-3 + CD4 + T cells and dCTLA-4 − Tim-3 − CD4 + T cells. n = 9. c Expression of T-bet, GATA-3, Foxp3, and ROR-γt of dCTLA-4 + Tim-3 + CD4 + T cells and dCTLA-4 - Tim-3 - CD4 + T cells. n = 9, Data represented the mean ± SEM. The flow cytometry plots were representative of three independent experiments. * P
Figure Legend Snippet: Cytokine production in dCD4 + T cells during normal pregnancy. a Production of the pro-inflammatory cytokines TNF-α, IFN-γ and IL-17A in dCTLA-4 + Tim-3 + CD4 + T cells and dCTLA-4 − Tim-3 − CD4 + T cells from the first trimester of human normal pregnancies. n = 9. b Quantitation of flow cytometric analysis of IL-4, TGF-β1, and IL-10 of dCTLA-4 + Tim-3 + CD4 + T cells and dCTLA-4 − Tim-3 − CD4 + T cells. n = 9. c Expression of T-bet, GATA-3, Foxp3, and ROR-γt of dCTLA-4 + Tim-3 + CD4 + T cells and dCTLA-4 - Tim-3 - CD4 + T cells. n = 9, Data represented the mean ± SEM. The flow cytometry plots were representative of three independent experiments. * P

Techniques Used: Quantitation Assay, Flow Cytometry, Expressing, Cytometry

Effect of blocking CTLA-4 and Tim-3 signaling pathways on cytokines production by human dCD4 + T cells. a Expression of Th1-type cytokines and transcription factors of dCD4 + T cells cultured for 48 h in the presence or absence of anti-CTLA-4 antibody (10 μg/ml), anti-Tim-3 antibody (10 μg/ml), or both. b Quantification of flow cytometric analysis of IL-4 and GATA-3 expression by dCD4 + T cells following treatment with the indicated blocking antibodies. c Expression of Treg-type cytokines by dCD4 + T cells following treatment with the indicated blocking antibodies. Data represented the mean ± SEM. n = 12. * P
Figure Legend Snippet: Effect of blocking CTLA-4 and Tim-3 signaling pathways on cytokines production by human dCD4 + T cells. a Expression of Th1-type cytokines and transcription factors of dCD4 + T cells cultured for 48 h in the presence or absence of anti-CTLA-4 antibody (10 μg/ml), anti-Tim-3 antibody (10 μg/ml), or both. b Quantification of flow cytometric analysis of IL-4 and GATA-3 expression by dCD4 + T cells following treatment with the indicated blocking antibodies. c Expression of Treg-type cytokines by dCD4 + T cells following treatment with the indicated blocking antibodies. Data represented the mean ± SEM. n = 12. * P

Techniques Used: Blocking Assay, Expressing, Cell Culture, Flow Cytometry

15) Product Images from "Selective ORAI1 inhibition ameliorates autoimmune CNS inflammation by suppressing effector but not regulatory T cell function"

Article Title: Selective ORAI1 inhibition ameliorates autoimmune CNS inflammation by suppressing effector but not regulatory T cell function

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1501406

Progression and severity of EAE are attenuated by treatment of mice with CRAC channel inhibitor ( A ) Clinical EAE score in WT mice injected with MOG 35-55 in CFA. Mice were treated with 6 mg/kg of the CRAC channel inhibitor AMG1 or vehicle control for 10 days starting when EAE scores were ≥ 1. Graphs represent the average ± SEM of 8 mice per group. ( B ) Ca 2+ influx was measured in splenocytes isolated at the end of 10 day treatment with inhibitor or vehicle. Cells were loaded with Fura2-AM and stimulated with 1 μM thapsigargin (TG) in Ca 2+ free buffer, followed by readdition of 1 mM extracellular Ca 2+ to induce SOCE. ( C ) Absolute numbers of CD4 + and CD8 + T cells and CD11b + Gr-1 + polymorphonuclear (PMN) cells, CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - DC isolated from the CNS of mice at day 23 after EAE induction and analyzed by flow cytometry. ( D ) Frequencies of CD4 + Foxp3 + Treg cells in the CNS at day 23 after EAE induction. ( E ) Expression of IFN-γ, IL-17 and GM-CSF by CD4 + T cells isolated from the CNS at day 23 after EAE induction and stimulated with PMA and ionomycin for 6 h. Data represent the average ± SEM of 8 mice per group. Statistical analysis of EAE scores in A (days 13–23) was performed using a Mann-Whitney test; data in panels B–E were analyzed using an unpaired Student’s t test. * p
Figure Legend Snippet: Progression and severity of EAE are attenuated by treatment of mice with CRAC channel inhibitor ( A ) Clinical EAE score in WT mice injected with MOG 35-55 in CFA. Mice were treated with 6 mg/kg of the CRAC channel inhibitor AMG1 or vehicle control for 10 days starting when EAE scores were ≥ 1. Graphs represent the average ± SEM of 8 mice per group. ( B ) Ca 2+ influx was measured in splenocytes isolated at the end of 10 day treatment with inhibitor or vehicle. Cells were loaded with Fura2-AM and stimulated with 1 μM thapsigargin (TG) in Ca 2+ free buffer, followed by readdition of 1 mM extracellular Ca 2+ to induce SOCE. ( C ) Absolute numbers of CD4 + and CD8 + T cells and CD11b + Gr-1 + polymorphonuclear (PMN) cells, CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - DC isolated from the CNS of mice at day 23 after EAE induction and analyzed by flow cytometry. ( D ) Frequencies of CD4 + Foxp3 + Treg cells in the CNS at day 23 after EAE induction. ( E ) Expression of IFN-γ, IL-17 and GM-CSF by CD4 + T cells isolated from the CNS at day 23 after EAE induction and stimulated with PMA and ionomycin for 6 h. Data represent the average ± SEM of 8 mice per group. Statistical analysis of EAE scores in A (days 13–23) was performed using a Mann-Whitney test; data in panels B–E were analyzed using an unpaired Student’s t test. * p

Techniques Used: Mouse Assay, Injection, Isolation, Flow Cytometry, Cytometry, Expressing, MANN-WHITNEY

Inducible deletion of Orai1 gene in T cells during ongoing EAE ameliorates disease severity ( A,B ) In vitro deletion of Orai1 in CD4 + T cells isolated from Orai1 fl/fl Cre-ERT2 , WT and Orai1 fl/fl Cd4-Cre control mice. Cells were stimulated with anti-CD3/CD28 for 3 days in the presence of 1 μM tamoxifen (TAM) and analyzed for SOCE after stimulation with thapsigargin and addition of 1 mM Ca 2+ ( A ) and Orai1 mRNA expression by RT PCR ( B ). ( C–F ) For passive induction of EAE, Orai1 fl/fl Cre-ERT2 and WT mice were immunized with MOG peptide. T cells were isolated from spleen and LNs 12 days later and restimulated in vitro with MOG peptide in the presence of IL-23 for 3 days. 4 × 10 6 CD4 + T cells were transferred i.v. into sublethally irradiated CD45.1 recipient mice. After EAE symptoms developed, recipient mice were injected with tamoxifen or vehicle control from day 10–14 after T cell transfer. ( C ) Clinical EAE scores. ( D–F ) Absolute numbers of CD4 + and CD8 + T cells ( D ) as well as CD11b + Gr-1 + polymorphonuclear cells (PMN), CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - dendritic cells ( E ) in the CNS 18 days after adoptive transfer. ( F ) Frequencies of CD4 + IFN-γ + and CD4 + IL-17 + T cells isolated from the CNS and restimulated in vitro with PMA and ionomycin for 6h. Data represent the average ± SEM of 3–4 mice per group. Statistical analysis of Orai1 expression in B was performed using a one-way ANOVA test. Statistical analysis of EAE scores (days 10–18) in C was performed using a Mann-Whitney test. Data in panels D–F were analyzed using an unpaired Student’s t test. * p
Figure Legend Snippet: Inducible deletion of Orai1 gene in T cells during ongoing EAE ameliorates disease severity ( A,B ) In vitro deletion of Orai1 in CD4 + T cells isolated from Orai1 fl/fl Cre-ERT2 , WT and Orai1 fl/fl Cd4-Cre control mice. Cells were stimulated with anti-CD3/CD28 for 3 days in the presence of 1 μM tamoxifen (TAM) and analyzed for SOCE after stimulation with thapsigargin and addition of 1 mM Ca 2+ ( A ) and Orai1 mRNA expression by RT PCR ( B ). ( C–F ) For passive induction of EAE, Orai1 fl/fl Cre-ERT2 and WT mice were immunized with MOG peptide. T cells were isolated from spleen and LNs 12 days later and restimulated in vitro with MOG peptide in the presence of IL-23 for 3 days. 4 × 10 6 CD4 + T cells were transferred i.v. into sublethally irradiated CD45.1 recipient mice. After EAE symptoms developed, recipient mice were injected with tamoxifen or vehicle control from day 10–14 after T cell transfer. ( C ) Clinical EAE scores. ( D–F ) Absolute numbers of CD4 + and CD8 + T cells ( D ) as well as CD11b + Gr-1 + polymorphonuclear cells (PMN), CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - dendritic cells ( E ) in the CNS 18 days after adoptive transfer. ( F ) Frequencies of CD4 + IFN-γ + and CD4 + IL-17 + T cells isolated from the CNS and restimulated in vitro with PMA and ionomycin for 6h. Data represent the average ± SEM of 3–4 mice per group. Statistical analysis of Orai1 expression in B was performed using a one-way ANOVA test. Statistical analysis of EAE scores (days 10–18) in C was performed using a Mann-Whitney test. Data in panels D–F were analyzed using an unpaired Student’s t test. * p

Techniques Used: In Vitro, Isolation, Mouse Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Irradiation, Injection, Adoptive Transfer Assay, MANN-WHITNEY

Experimental autoimmune encephalomyelitis (EAE) is ameliorated in Orai1 fl/fl Cd4-Cre mice ( A ) RNA from wildtype and Orai1 fl/fl Cd4-Cre was isolated from CD4 + T cells and relative mRNA expression was measured by RT PCR. ( B ) SOCE was measured in Fura2-AM loaded freshly isolated CD4 + T cells from wildtype and Orai1 fl/fl Cd4-Cre mice after stimulation with thapsigargin (TG) followed by readdition of 1 mM Ca 2+ to the extracellular medium. ( C ) Clinical EAE scores in WT and Orai1 fl/fl Cd4-Cre mice injected with MOG 35-55 in CFA. ( D ) Summary of EAE with days to onset (±SEM), incidence of EAE and maximal EAE scores (±SEM). ( E ) H E and Luxol fast blue staining of spinal cords from WT and Orai1 fl/fl Cd4-Cre mice at 27 after EAE induction. Arrows indicate infiltrating cells and demyelination, respectively. Shown is one histological stain representative of 4 mice per group and 6 examined levels of spinal cord per mouse. ( F–I ) Cells were isolated from the spinal cord (CNS) of WT and Orai1 fl/fl Cd4-Cre mice at day 27 after EAE induction and analyzed by flow cytometry. ( F–H ) Absolute numbers of CD4 + and CD8 + T cells ( F ), frequencies of Foxp3 + CD4 + T cells ( G ) and absolute numbers of CD11b + Gr-1 + polymorphonuclear cells (PMN), CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - dendritic cells in the CNS ( H ). ( I ) Cytokine production of T cells isolated from the CNS was analyzed by flow cytometry after stimulation with PMA and ionomycin in the presence of Brefeldin A for 6 h. Shown are absolute numbers of CD4 + T cells expressing IFN-γ, IL-17 or GM-CSF. Data in panels A and B represent the average ± SEM of 3 mice per group. Data in panels C–D and F–H represent the average ± SEM of 14–16 mice per group. Statistical analysis of EAE scores (starting at the first clinical signs of EAE) was performed using a Mann-Whitney test (panel C); data in panels B, F–H were analyzed using an unpaired Student’s t test. * p
Figure Legend Snippet: Experimental autoimmune encephalomyelitis (EAE) is ameliorated in Orai1 fl/fl Cd4-Cre mice ( A ) RNA from wildtype and Orai1 fl/fl Cd4-Cre was isolated from CD4 + T cells and relative mRNA expression was measured by RT PCR. ( B ) SOCE was measured in Fura2-AM loaded freshly isolated CD4 + T cells from wildtype and Orai1 fl/fl Cd4-Cre mice after stimulation with thapsigargin (TG) followed by readdition of 1 mM Ca 2+ to the extracellular medium. ( C ) Clinical EAE scores in WT and Orai1 fl/fl Cd4-Cre mice injected with MOG 35-55 in CFA. ( D ) Summary of EAE with days to onset (±SEM), incidence of EAE and maximal EAE scores (±SEM). ( E ) H E and Luxol fast blue staining of spinal cords from WT and Orai1 fl/fl Cd4-Cre mice at 27 after EAE induction. Arrows indicate infiltrating cells and demyelination, respectively. Shown is one histological stain representative of 4 mice per group and 6 examined levels of spinal cord per mouse. ( F–I ) Cells were isolated from the spinal cord (CNS) of WT and Orai1 fl/fl Cd4-Cre mice at day 27 after EAE induction and analyzed by flow cytometry. ( F–H ) Absolute numbers of CD4 + and CD8 + T cells ( F ), frequencies of Foxp3 + CD4 + T cells ( G ) and absolute numbers of CD11b + Gr-1 + polymorphonuclear cells (PMN), CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - dendritic cells in the CNS ( H ). ( I ) Cytokine production of T cells isolated from the CNS was analyzed by flow cytometry after stimulation with PMA and ionomycin in the presence of Brefeldin A for 6 h. Shown are absolute numbers of CD4 + T cells expressing IFN-γ, IL-17 or GM-CSF. Data in panels A and B represent the average ± SEM of 3 mice per group. Data in panels C–D and F–H represent the average ± SEM of 14–16 mice per group. Statistical analysis of EAE scores (starting at the first clinical signs of EAE) was performed using a Mann-Whitney test (panel C); data in panels B, F–H were analyzed using an unpaired Student’s t test. * p

Techniques Used: Mouse Assay, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Injection, Staining, Flow Cytometry, Cytometry, MANN-WHITNEY

16) Product Images from "T cell restricted Notch signaling contributes to pulmonary Th1 and Th2 immunity during Cryptococcus neoformans infection"

Article Title: T cell restricted Notch signaling contributes to pulmonary Th1 and Th2 immunity during Cryptococcus neoformans infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1601715

Inhibition of Notch signaling reduces frequencies of Th1 and Th2 cells in the lungs during C. neoformans infection Lung leukocytes were isolated from perfused CCD and WT mice at 4 wpi. (A) Cells were stimulated with plate bound anti-CD3 and anti-CD28 antibodies and analyzed for the proportion of cells producing (A) IFN-γ, (B) IL-5, (C) IL-13 and (D) IL-17A by flow cytometry. The geometric mean fluorescence intensity (MFI) of the positive populations is quantified next to each plot. (E) Expression of Tbet was quantified in lung leukocytes by qRT-PCR and (F) within lung CD4 + T cells by flow cytometry. (G) Expression of Gata3 was quantified in lung leukocytes by qRT-PCR and (H) within lung CD4+ T cells by flow cytometry. (I) The frequency of FoxP3 + CD4 + Treg was determined by flow cytometry and (J) expression of Foxp3 in the lugs was determined by qRT-PCR. Flow cytometry plots shown are gated on Live, CD45 + , TCRβ + CD4 + T cells and are representative examples of 1-4 independent experiments. FMO controls were used to set cytokine gates. Frequencies and MFI (geometric mean) data shown are the mean ± SEM with n=5-7/group. Relative gene expression is normalized to Gapdh . Data shown are the mean ± SEM of n=3-7/group. *p
Figure Legend Snippet: Inhibition of Notch signaling reduces frequencies of Th1 and Th2 cells in the lungs during C. neoformans infection Lung leukocytes were isolated from perfused CCD and WT mice at 4 wpi. (A) Cells were stimulated with plate bound anti-CD3 and anti-CD28 antibodies and analyzed for the proportion of cells producing (A) IFN-γ, (B) IL-5, (C) IL-13 and (D) IL-17A by flow cytometry. The geometric mean fluorescence intensity (MFI) of the positive populations is quantified next to each plot. (E) Expression of Tbet was quantified in lung leukocytes by qRT-PCR and (F) within lung CD4 + T cells by flow cytometry. (G) Expression of Gata3 was quantified in lung leukocytes by qRT-PCR and (H) within lung CD4+ T cells by flow cytometry. (I) The frequency of FoxP3 + CD4 + Treg was determined by flow cytometry and (J) expression of Foxp3 in the lugs was determined by qRT-PCR. Flow cytometry plots shown are gated on Live, CD45 + , TCRβ + CD4 + T cells and are representative examples of 1-4 independent experiments. FMO controls were used to set cytokine gates. Frequencies and MFI (geometric mean) data shown are the mean ± SEM with n=5-7/group. Relative gene expression is normalized to Gapdh . Data shown are the mean ± SEM of n=3-7/group. *p

Techniques Used: Inhibition, Infection, Isolation, Mouse Assay, Flow Cytometry, Cytometry, Fluorescence, Expressing, Quantitative RT-PCR

Inhibition of Notch signaling impairs IFN-γ production by CD8 + T cells in the lungs during C. neoformans infection Lung leukocytes were isolated from perfused CCD and WT mice at 4 wpi. (A) The total number of CD8 + T cells were quantified and (B) CD44 and CD62L staining was used to assess activation and effector phenotype after gating on Live, CD45 + TCRβ+ CD8+ cells by flow cytometry. (C) Lung leukocytes were stimulated with plate bound anti-CD3 and anti-CD28 antibodies and analyzed for the proportion of CD8 + T cells producing IFN-γ and (D) expression of Eomesodermin was determined by flow cytometry. Flow cytometry plots shown are gated on Live, CD45 + , TCRβ + CD8 + T cells and are representative examples of 1-4 independent experiments. FMO controls were used to set cytokine gates. Frequencies and MFI (geometric mean) data shown are the mean ± SEM with n=5-7/group. *p
Figure Legend Snippet: Inhibition of Notch signaling impairs IFN-γ production by CD8 + T cells in the lungs during C. neoformans infection Lung leukocytes were isolated from perfused CCD and WT mice at 4 wpi. (A) The total number of CD8 + T cells were quantified and (B) CD44 and CD62L staining was used to assess activation and effector phenotype after gating on Live, CD45 + TCRβ+ CD8+ cells by flow cytometry. (C) Lung leukocytes were stimulated with plate bound anti-CD3 and anti-CD28 antibodies and analyzed for the proportion of CD8 + T cells producing IFN-γ and (D) expression of Eomesodermin was determined by flow cytometry. Flow cytometry plots shown are gated on Live, CD45 + , TCRβ + CD8 + T cells and are representative examples of 1-4 independent experiments. FMO controls were used to set cytokine gates. Frequencies and MFI (geometric mean) data shown are the mean ± SEM with n=5-7/group. *p

Techniques Used: Inhibition, Infection, Isolation, Mouse Assay, Staining, Activation Assay, Flow Cytometry, Cytometry, Expressing

17) Product Images from "CD5-dependent CK2 activation pathway regulates threshold for T-cell anergy"

Article Title: CD5-dependent CK2 activation pathway regulates threshold for T-cell anergy

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1200065

Contribution of the CD5-CK2 signaling pathway to the generation of Th1, Th2, and Th17 cells from naïve CD4 + T-cells. Naïve CD4 + T-cells were co-cultured with irradiated APCs and stimulated with MOG 35-55 peptide ( A ) or anti-CD3 ( B ) under non-polarizing (unpolarized) or polarizing conditions and generation of Th subpopulations was evaluated. Numbers in each quadrant represent the average frequency of CD4 + T-cells expressing IFNγ, IL-17a and/or IL-4±SEM from four independent experiments performed with at least three mice per experiment. C , Percent 7-AAD + CD4 + T-cell at 24 h and 120 h and D , RORγt + CD4 + T-cells at 120 h following stimulation with MOG 35-55 peptide under Th17 polarizing conditions. Data is mean±SEM of three independent experiments with at least two mice/group. E , pSTAT1 (Ser727) and F , pSTAT3 (Tyr705) in CD4+ T-cells stimulated for 72h with MOG 35-55 peptide under Th17 polarizing conditions. The filled shaded histograms are unstimulated and the dashed histograms are isotype immunoglobulin stained cell controls. For pSTAT1 ( E ), the MFI of 2D2.CD5ΔCK2BD is 52±4.8 and 2D2.CD5WT is 30±4.3. For pSTAT3 ( F ) the MFI for 2D2.CD5ΔCK2BD is 32±2.9 and 2D2.CD5WT is 21±3.5. Histograms represent data from one representative mouse per group from two mice/experiment, n=3 experiments. *p
Figure Legend Snippet: Contribution of the CD5-CK2 signaling pathway to the generation of Th1, Th2, and Th17 cells from naïve CD4 + T-cells. Naïve CD4 + T-cells were co-cultured with irradiated APCs and stimulated with MOG 35-55 peptide ( A ) or anti-CD3 ( B ) under non-polarizing (unpolarized) or polarizing conditions and generation of Th subpopulations was evaluated. Numbers in each quadrant represent the average frequency of CD4 + T-cells expressing IFNγ, IL-17a and/or IL-4±SEM from four independent experiments performed with at least three mice per experiment. C , Percent 7-AAD + CD4 + T-cell at 24 h and 120 h and D , RORγt + CD4 + T-cells at 120 h following stimulation with MOG 35-55 peptide under Th17 polarizing conditions. Data is mean±SEM of three independent experiments with at least two mice/group. E , pSTAT1 (Ser727) and F , pSTAT3 (Tyr705) in CD4+ T-cells stimulated for 72h with MOG 35-55 peptide under Th17 polarizing conditions. The filled shaded histograms are unstimulated and the dashed histograms are isotype immunoglobulin stained cell controls. For pSTAT1 ( E ), the MFI of 2D2.CD5ΔCK2BD is 52±4.8 and 2D2.CD5WT is 30±4.3. For pSTAT3 ( F ) the MFI for 2D2.CD5ΔCK2BD is 32±2.9 and 2D2.CD5WT is 21±3.5. Histograms represent data from one representative mouse per group from two mice/experiment, n=3 experiments. *p

Techniques Used: Cell Culture, Irradiation, Expressing, Mouse Assay, Staining

CD5-CK2 signaling pathway-deficient T-cells are resistant to development of anergy. Purified CD4 + T-cells were stimulated for 24 h with anti-CD3 or MOG 35-55 peptide, rested for 72 h, and then re-stimulated for 48 h with anti-CD3 or MOG 35-55 peptide. A and B , Frequency of CD4 + T-cells incorporating [ 3 H]thymidine following 48 h re-stimulation with varying concentrations of anti-CD3 ( A ) or MOG 35-55 peptide ( B ). Data ± SEM represent the average of 2 mice per group per experiment, n = 3 experiments. C and D , EdU incorporation by CD4 + T-cells restimulated with anti-CD3( C ) or MOG 35-55 peptide ( D ). Cells were pulse labeled with EdU after 48 h re-stimulation. E , EdU incorporation in CD8 + T-cells 48 h after re-stimulation with 1 μg/ml anti-CD3. F and G , EdU incorporation in co-cultures of F , CFSE-labeled CD5WT and unlabeled CD5ΔCK2BD CD4 + T-cells restimulated with anti-CD3 mAb or G , CFSE-labeled 2D2.CD5WT and unlabeled 2D2.ΔCK2BD CD4 + T-cells restimulated with MOG 35-55 for the indicated times. H , Effect of in vitro restimulation on in vivo primed T-cells. CD5WT mice and CD5ΔCK2BD mice were immunized with 150 μg MOG 35-55 peptide in CFA, s.c. Seven days later CD4 + T-cells from spleens from both groups of mice were co-cultured in the presence of MOG 35-55 peptide and proliferation was assessed by EdU incorporation at the indicated time points. Data ± SEM represent the average of 2-3 mice per group per experiment, n = at least 3 experiments. Bars above plots denote regions of statistical significance. * p
Figure Legend Snippet: CD5-CK2 signaling pathway-deficient T-cells are resistant to development of anergy. Purified CD4 + T-cells were stimulated for 24 h with anti-CD3 or MOG 35-55 peptide, rested for 72 h, and then re-stimulated for 48 h with anti-CD3 or MOG 35-55 peptide. A and B , Frequency of CD4 + T-cells incorporating [ 3 H]thymidine following 48 h re-stimulation with varying concentrations of anti-CD3 ( A ) or MOG 35-55 peptide ( B ). Data ± SEM represent the average of 2 mice per group per experiment, n = 3 experiments. C and D , EdU incorporation by CD4 + T-cells restimulated with anti-CD3( C ) or MOG 35-55 peptide ( D ). Cells were pulse labeled with EdU after 48 h re-stimulation. E , EdU incorporation in CD8 + T-cells 48 h after re-stimulation with 1 μg/ml anti-CD3. F and G , EdU incorporation in co-cultures of F , CFSE-labeled CD5WT and unlabeled CD5ΔCK2BD CD4 + T-cells restimulated with anti-CD3 mAb or G , CFSE-labeled 2D2.CD5WT and unlabeled 2D2.ΔCK2BD CD4 + T-cells restimulated with MOG 35-55 for the indicated times. H , Effect of in vitro restimulation on in vivo primed T-cells. CD5WT mice and CD5ΔCK2BD mice were immunized with 150 μg MOG 35-55 peptide in CFA, s.c. Seven days later CD4 + T-cells from spleens from both groups of mice were co-cultured in the presence of MOG 35-55 peptide and proliferation was assessed by EdU incorporation at the indicated time points. Data ± SEM represent the average of 2-3 mice per group per experiment, n = at least 3 experiments. Bars above plots denote regions of statistical significance. * p

Techniques Used: Purification, Mouse Assay, Labeling, In Vitro, In Vivo, Cell Culture

18) Product Images from "A critical role of IL-17 in modulating the B-cell response during H5N1 influenza virus infection"

Article Title: A critical role of IL-17 in modulating the B-cell response during H5N1 influenza virus infection

Journal: Cellular and Molecular Immunology

doi: 10.1038/cmi.2011.38

The total number and frequency of B cells were significantly reduced in the lung tissue of IL-17 KO mice upon virus stimulation. ( a ) Immunohistochemical staining of B220 + B cells in the lungs collected at 5 dpi. Sections are representative of four mice in each group. Images are at magnification ×400. ( b ) Flow cytometric analysis of B-cell frequencies among immune cells in the lung 48 h post-stimulation with inactivated virus. Numbers indicate the percentage of B220 + cells. Figures are representative of three mice in each group. ( c, d ) Cellularity analysis of B cells (B220 + ), T cells (CD3 + ), γσT cells (γσTCR + ), NK cells (NK1.1 + CD3 - ), NKT cells (NK1.1 + CD3 + ), DCs (CD11c + ), neutrophils (Gr1 + CD11b + ) and macrophages (Gr1 − CD11b + ) in the lungs 48 h post-stimulation with inactivated virus ( n =3 in each group). * P
Figure Legend Snippet: The total number and frequency of B cells were significantly reduced in the lung tissue of IL-17 KO mice upon virus stimulation. ( a ) Immunohistochemical staining of B220 + B cells in the lungs collected at 5 dpi. Sections are representative of four mice in each group. Images are at magnification ×400. ( b ) Flow cytometric analysis of B-cell frequencies among immune cells in the lung 48 h post-stimulation with inactivated virus. Numbers indicate the percentage of B220 + cells. Figures are representative of three mice in each group. ( c, d ) Cellularity analysis of B cells (B220 + ), T cells (CD3 + ), γσT cells (γσTCR + ), NK cells (NK1.1 + CD3 - ), NKT cells (NK1.1 + CD3 + ), DCs (CD11c + ), neutrophils (Gr1 + CD11b + ) and macrophages (Gr1 − CD11b + ) in the lungs 48 h post-stimulation with inactivated virus ( n =3 in each group). * P

Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Flow Cytometry

19) Product Images from "A novel mechanism for generating the interferon signature in lupus: opsonization of dead cells by complement and IgM"

Article Title: A novel mechanism for generating the interferon signature in lupus: opsonization of dead cells by complement and IgM

Journal: Arthritis & rheumatology (Hoboken, N.J.)

doi: 10.1002/art.39781

Cytokine production is C3-dependent Wild type (WT) and C3−/− mice were treated with pristane, left untreated, or given cobra venom factor (CVF) on day 0 and 7 following pristane treatment. A, Left, expression of Mx1 relative to 18S rRNA (real-time PCR). Right, expression of Sca-1 on B220 + B cells (flow cytometry). B, Percentages of Ly6G + intracellular TNFα + neutrophils in peritoneal exudate cells (PECs) from WT or C3−/− mice 2-wks after pristane treatment and in WT mice treated with CVF. C and D, TNFα (C) and IL-12 (D) levels in peritoneal lavage fluid from WT or C3−/− mice 0–4 weeks after pristane treatment (ELISA). E, Sca-1 protein expression on B220 + B cells by flow cytometry (left) and expression of Mx1 relative to 18S rRNA (real-time PCR) in CD18−/− vs. WT mice (* P
Figure Legend Snippet: Cytokine production is C3-dependent Wild type (WT) and C3−/− mice were treated with pristane, left untreated, or given cobra venom factor (CVF) on day 0 and 7 following pristane treatment. A, Left, expression of Mx1 relative to 18S rRNA (real-time PCR). Right, expression of Sca-1 on B220 + B cells (flow cytometry). B, Percentages of Ly6G + intracellular TNFα + neutrophils in peritoneal exudate cells (PECs) from WT or C3−/− mice 2-wks after pristane treatment and in WT mice treated with CVF. C and D, TNFα (C) and IL-12 (D) levels in peritoneal lavage fluid from WT or C3−/− mice 0–4 weeks after pristane treatment (ELISA). E, Sca-1 protein expression on B220 + B cells by flow cytometry (left) and expression of Mx1 relative to 18S rRNA (real-time PCR) in CD18−/− vs. WT mice (* P

Techniques Used: Mouse Assay, Combined Bisulfite Restriction Analysis Assay, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

In vivo phagocytosis of apoptotic cells is C3-dependent A , flow cytometry analysis of the uptake of pHrodo red-labeled apoptotic BW5147 cells after i.p. injection into pristane-treated wild type (WT) or C3−/− mice or WT mice treated with cobra venom factor (CVF) at day 0 and 7 after pristane treatment. B, in vivo phagocytosis of pHrodo red-labeled apoptotic BW5147 cells by CD11b + cells. C, flow cytometry showing uptake of pHrodo red-labeled apoptotic cells by macrophages (MΦ), monocytes (Mono), and neutrophils (Neut). D, WT and C3−/− mice were treated with pristane, left untreated, or given CVF on day 0 and 7 following pristane treatment and PECs were analyzed by flow cytometry 2-wks later. E, Total peritoneal exudate cell counts. F, Percentages of Ly6C low macrophages, Ly6C hi monocytes, and Ly6G + neutrophils. G, Percentages of Tim4 + CD11b + resident macrophages. (* P
Figure Legend Snippet: In vivo phagocytosis of apoptotic cells is C3-dependent A , flow cytometry analysis of the uptake of pHrodo red-labeled apoptotic BW5147 cells after i.p. injection into pristane-treated wild type (WT) or C3−/− mice or WT mice treated with cobra venom factor (CVF) at day 0 and 7 after pristane treatment. B, in vivo phagocytosis of pHrodo red-labeled apoptotic BW5147 cells by CD11b + cells. C, flow cytometry showing uptake of pHrodo red-labeled apoptotic cells by macrophages (MΦ), monocytes (Mono), and neutrophils (Neut). D, WT and C3−/− mice were treated with pristane, left untreated, or given CVF on day 0 and 7 following pristane treatment and PECs were analyzed by flow cytometry 2-wks later. E, Total peritoneal exudate cell counts. F, Percentages of Ly6C low macrophages, Ly6C hi monocytes, and Ly6G + neutrophils. G, Percentages of Tim4 + CD11b + resident macrophages. (* P

Techniques Used: In Vivo, Flow Cytometry, Cytometry, Labeling, Injection, Mouse Assay, Combined Bisulfite Restriction Analysis Assay

20) Product Images from "Integrin αvβ3 enhances the suppressive effect of interferon‐γ on hematopoietic stem cells"

Article Title: Integrin αvβ3 enhances the suppressive effect of interferon‐γ on hematopoietic stem cells

Journal: The EMBO Journal

doi: 10.15252/embj.201796771

Expression of integrin β3 contributes to the accurate identification of HSCs after IFNγ treatment A After WT mice were intravenously injected with 1 μg IFNγ three times every 48 h, the frequency of LT‐HSC fraction (CD150 + CD34 − KSL) was examined. The graph depicts the frequency of LT‐HSC fraction. Data are presented as means ± SD, and were analyzed using Student's t ‐test ( n = 5, * P
Figure Legend Snippet: Expression of integrin β3 contributes to the accurate identification of HSCs after IFNγ treatment A After WT mice were intravenously injected with 1 μg IFNγ three times every 48 h, the frequency of LT‐HSC fraction (CD150 + CD34 − KSL) was examined. The graph depicts the frequency of LT‐HSC fraction. Data are presented as means ± SD, and were analyzed using Student's t ‐test ( n = 5, * P

Techniques Used: Expressing, Mouse Assay, Injection

Integrin β3 signaling is involved in the suppressive effect of IFNγ on HSC pool A, B After WT and integrin β3 Y747A (Y747A) mice were intravenously injected with 1 μg IFNγ three times every 48 h, the frequency or absolute number of integrin β3 High CD150 + CD34 − c‐kit + Lineage − (integrin β3 High CD150 + CD34 − KL) cells was examined. The graph depicts the frequency (A) or absolute number (B) of integrin β3 High CD150 + CD34 − KL cells in WT or Y747A mice after PBS or IFN γ administration. Data are presented as means ± SD, and were analyzed using Student's t ‐test ( n = 5, ** P
Figure Legend Snippet: Integrin β3 signaling is involved in the suppressive effect of IFNγ on HSC pool A, B After WT and integrin β3 Y747A (Y747A) mice were intravenously injected with 1 μg IFNγ three times every 48 h, the frequency or absolute number of integrin β3 High CD150 + CD34 − c‐kit + Lineage − (integrin β3 High CD150 + CD34 − KL) cells was examined. The graph depicts the frequency (A) or absolute number (B) of integrin β3 High CD150 + CD34 − KL cells in WT or Y747A mice after PBS or IFN γ administration. Data are presented as means ± SD, and were analyzed using Student's t ‐test ( n = 5, ** P

Techniques Used: Mouse Assay, Injection

Integrin β3 signaling promotes IFNγ‐dependent suppression of HSC function After 50 CD150 + CD34 − KSL LT‐HSCs derived from WT mice (Ly5.1) were sorted and cultured for 5 days on plates with or without of vitronectin (VN) coating, in the presence of both SCF and TPO, with or without IFNγ, whole cultured cells were transplanted into lethally irradiated mice (Ly5.2) along with 5 × 10 5 BM competitor cells (Ly5.2). Twenty weeks later, the % donor cells (Ly5.1 + ) were determined in peripheral blood (1 st ). Then, 12 weeks after secondary transplantation with chimeric BM cells from the primary recipients, peripheral blood from the secondary recipients was analyzed (2 nd ). Recipient mice with
Figure Legend Snippet: Integrin β3 signaling promotes IFNγ‐dependent suppression of HSC function After 50 CD150 + CD34 − KSL LT‐HSCs derived from WT mice (Ly5.1) were sorted and cultured for 5 days on plates with or without of vitronectin (VN) coating, in the presence of both SCF and TPO, with or without IFNγ, whole cultured cells were transplanted into lethally irradiated mice (Ly5.2) along with 5 × 10 5 BM competitor cells (Ly5.2). Twenty weeks later, the % donor cells (Ly5.1 + ) were determined in peripheral blood (1 st ). Then, 12 weeks after secondary transplantation with chimeric BM cells from the primary recipients, peripheral blood from the secondary recipients was analyzed (2 nd ). Recipient mice with

Techniques Used: Derivative Assay, Mouse Assay, Cell Culture, Irradiation, Transplantation Assay

Integrin β3 signaling supports the effect of IFNγ through STAT1 STAT1 −/− CD150 + CD34 − KSL HSCs (Ly5.2) were cultured for 5 days in the presence of SCF and TPO, with or without vitronectin (VN), in the absence or presence of IFNγ, after which they were transplanted into lethally irradiated mice (Ly5.1) along with 5 × 10 5 BM competitor cells (Ly5.1). Twenty weeks later, the percent donor cells (Ly5.2 + ) were determined in peripheral blood. Each plot depicts the chimerism of donor‐derived cells (% Ly5.2 + cells) in the peripheral blood of recipient mice. Bars indicate mean values. Data were analyzed using Student's t ‐test ( n = 11−14, * P
Figure Legend Snippet: Integrin β3 signaling supports the effect of IFNγ through STAT1 STAT1 −/− CD150 + CD34 − KSL HSCs (Ly5.2) were cultured for 5 days in the presence of SCF and TPO, with or without vitronectin (VN), in the absence or presence of IFNγ, after which they were transplanted into lethally irradiated mice (Ly5.1) along with 5 × 10 5 BM competitor cells (Ly5.1). Twenty weeks later, the percent donor cells (Ly5.2 + ) were determined in peripheral blood. Each plot depicts the chimerism of donor‐derived cells (% Ly5.2 + cells) in the peripheral blood of recipient mice. Bars indicate mean values. Data were analyzed using Student's t ‐test ( n = 11−14, * P

Techniques Used: Cell Culture, Irradiation, Mouse Assay, Derivative Assay

21) Product Images from "Stem Cell Enrichment with Selectin Receptors: Mimicking the pH Environment of Trauma"

Article Title: Stem Cell Enrichment with Selectin Receptors: Mimicking the pH Environment of Trauma

Journal: Sensors (Basel, Switzerland)

doi: 10.3390/s130912516

Enhanced adhesion of CD34+ cells to L-selectin at acidic pH. ( A ) Relative fluorescence intensity of L-selectin coated and blank microtubes labeled with APC-anti human L-selectin. ( B – C ) Images of perfused cells interacting with blank or functionalized microtubes, respectively. Scale bars are 100 μm. ( D ) Rolling velocity of CD34+ cells under normal (7.4) and acidic (6.6) pH. CD34+ cells at a concentration of 1 × 10 6 cells/mL were perfused through L-selectin coated (20 μg/mL) microtubes at a shear stress of 2.0 dyn/cm 2 in buffer at specified pH. ( E ) Comparison of rolling velocities of CD34+ cells and MNCs (unpaired t-test, error bars indicate standard error of the mean; * p
Figure Legend Snippet: Enhanced adhesion of CD34+ cells to L-selectin at acidic pH. ( A ) Relative fluorescence intensity of L-selectin coated and blank microtubes labeled with APC-anti human L-selectin. ( B – C ) Images of perfused cells interacting with blank or functionalized microtubes, respectively. Scale bars are 100 μm. ( D ) Rolling velocity of CD34+ cells under normal (7.4) and acidic (6.6) pH. CD34+ cells at a concentration of 1 × 10 6 cells/mL were perfused through L-selectin coated (20 μg/mL) microtubes at a shear stress of 2.0 dyn/cm 2 in buffer at specified pH. ( E ) Comparison of rolling velocities of CD34+ cells and MNCs (unpaired t-test, error bars indicate standard error of the mean; * p

Techniques Used: Fluorescence, Labeling, Concentration Assay

L-selectin mediated isolation of CD34+ cells from patient bone marrow samples under acidic pH. Captured cells were labeled using a mouse anti-human CD34 monoclonal antibody. Flow cytometry plots are a representation of experiments done in triplicate. SSC = side scatter.
Figure Legend Snippet: L-selectin mediated isolation of CD34+ cells from patient bone marrow samples under acidic pH. Captured cells were labeled using a mouse anti-human CD34 monoclonal antibody. Flow cytometry plots are a representation of experiments done in triplicate. SSC = side scatter.

Techniques Used: Isolation, Labeling, Flow Cytometry, Cytometry

22) Product Images from "Maternal circulating leukocytes display early chemotactic responsiveness during late gestation"

Article Title: Maternal circulating leukocytes display early chemotactic responsiveness during late gestation

Journal: BMC Pregnancy and Childbirth

doi: 10.1186/1471-2393-13-S1-S8

Leukocyte subsets - maternal peripheral circulation. Leukocyte phenotype was determined by using monoclonal conjugated fluorochromes and flow cytometry. A, Total Leukocytes. B, T cell subsets. Data are presented as mean ± SEM of determinations in duplicate per group of tissues (n=5 each).
Figure Legend Snippet: Leukocyte subsets - maternal peripheral circulation. Leukocyte phenotype was determined by using monoclonal conjugated fluorochromes and flow cytometry. A, Total Leukocytes. B, T cell subsets. Data are presented as mean ± SEM of determinations in duplicate per group of tissues (n=5 each).

Techniques Used: Flow Cytometry, Cytometry

23) Product Images from "Clitocybe nuda Activates Dendritic Cells and Acts as a DNA Vaccine Adjuvant"

Article Title: Clitocybe nuda Activates Dendritic Cells and Acts as a DNA Vaccine Adjuvant

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2013/761454

WE-CN upregulates the expression of immunomodulatory cell surface markers on BMDCs. Immature BMDCs were stimulated with 100 ng/mL LPS or 100 μ g/mL WE-CN for 24 hr. The control group was treated with PBS alone. After incubation, the expression of the surface markers CD40, CD80, CD86, MHC class I, and MHC class II was analyzed by flow cytometry with fluorescently labeled Abs. All data were gated on CD11c + cells. The gray-filled area represents staining with an isotype-matched control Ab. (a) The histogram shows data from one representative experiment of each group. (b) The bar graphs represent the mean ± SD from three independent experiments.
Figure Legend Snippet: WE-CN upregulates the expression of immunomodulatory cell surface markers on BMDCs. Immature BMDCs were stimulated with 100 ng/mL LPS or 100 μ g/mL WE-CN for 24 hr. The control group was treated with PBS alone. After incubation, the expression of the surface markers CD40, CD80, CD86, MHC class I, and MHC class II was analyzed by flow cytometry with fluorescently labeled Abs. All data were gated on CD11c + cells. The gray-filled area represents staining with an isotype-matched control Ab. (a) The histogram shows data from one representative experiment of each group. (b) The bar graphs represent the mean ± SD from three independent experiments.

Techniques Used: Expressing, Incubation, Flow Cytometry, Cytometry, Labeling, Staining

24) Product Images from "Expression of the ARPC4 Subunit of Human Arp2/3 Severely Affects Mycobacterium tuberculosis Growth and Suppresses Immunogenic Response in Murine Macrophages"

Article Title: Expression of the ARPC4 Subunit of Human Arp2/3 Severely Affects Mycobacterium tuberculosis Growth and Suppresses Immunogenic Response in Murine Macrophages

Journal: PLoS ONE

doi: 10.1371/journal.pone.0069949

Activation of infected macrophages and inter-compartmental localization of mycobacteria within A. The plots depict reduced activation of H37RV/ARPC4 infected macrophages. a) Percentage of cells expressing PD1 among CD11B+ cells is shown in the dot plots with mean±STDEV. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains at 10 MOI, were surface-stained with anti-CD11B and PD1 antibodies and samples were acquired by flow cytometry. Data shown here are representative of three independent experiments. b) Cell death in H37Rv and H37Rv/ARPC4 Mtb infected macrophages. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains in 1∶10 ratio, were surface-stained with anti-CD11B, CD11C antibodies followed by PI staining for 20 minutes prior to acquisition by flow cytometry to assess cell death in infected macrophages. The percentage of cells expressing PI among CD11B±cells is shown with mean±STDEV. Data shown here are representative of three independent experiments. c) Expression of macrophage activation markers. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains at 10 MOI, were surface-stained with anti-CD11B, CD11C, MHCII, CD14, CD54, CD80, CD86 and CD69 antibodies and samples were acquired by flow cytometry. CD11B+ cells were gated for expression of MHCII, CD14, CD54, CD80 and CD86 and the percentage of cells expressing these markers are shown in the histogram overlay plots with mean±STDEV. Data shown here are representative of three independent experiments. B. Confocal Microscopy images showing that H37Rv/ARPC4 mycobacterial strain is less infectious and persistent in macrophage. FITC-labelled mycobacterial cells (green) were used for infecting macrophages (red) at an MOI of 5. At specific time-points, the cells were fixed, permeabilized and stained with anti-LAMP1 antibodies, followed by Alexa Fluor 594 goat anti-rat antibodies. (a)- (d) shows the infected macrophage cells at 0, 24, 48 and 72 hours' time-points post-infection, respectively. (e) and (f) are the graphical representation of the percentage of macrophage cells infected with FITC-labeled mycobacteria at different time-points and the percentage of bacteria localized in lysosomes in the infected cells, respectively. Experiments were repeated thrice and similar results were obtained.
Figure Legend Snippet: Activation of infected macrophages and inter-compartmental localization of mycobacteria within A. The plots depict reduced activation of H37RV/ARPC4 infected macrophages. a) Percentage of cells expressing PD1 among CD11B+ cells is shown in the dot plots with mean±STDEV. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains at 10 MOI, were surface-stained with anti-CD11B and PD1 antibodies and samples were acquired by flow cytometry. Data shown here are representative of three independent experiments. b) Cell death in H37Rv and H37Rv/ARPC4 Mtb infected macrophages. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains in 1∶10 ratio, were surface-stained with anti-CD11B, CD11C antibodies followed by PI staining for 20 minutes prior to acquisition by flow cytometry to assess cell death in infected macrophages. The percentage of cells expressing PI among CD11B±cells is shown with mean±STDEV. Data shown here are representative of three independent experiments. c) Expression of macrophage activation markers. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains at 10 MOI, were surface-stained with anti-CD11B, CD11C, MHCII, CD14, CD54, CD80, CD86 and CD69 antibodies and samples were acquired by flow cytometry. CD11B+ cells were gated for expression of MHCII, CD14, CD54, CD80 and CD86 and the percentage of cells expressing these markers are shown in the histogram overlay plots with mean±STDEV. Data shown here are representative of three independent experiments. B. Confocal Microscopy images showing that H37Rv/ARPC4 mycobacterial strain is less infectious and persistent in macrophage. FITC-labelled mycobacterial cells (green) were used for infecting macrophages (red) at an MOI of 5. At specific time-points, the cells were fixed, permeabilized and stained with anti-LAMP1 antibodies, followed by Alexa Fluor 594 goat anti-rat antibodies. (a)- (d) shows the infected macrophage cells at 0, 24, 48 and 72 hours' time-points post-infection, respectively. (e) and (f) are the graphical representation of the percentage of macrophage cells infected with FITC-labeled mycobacteria at different time-points and the percentage of bacteria localized in lysosomes in the infected cells, respectively. Experiments were repeated thrice and similar results were obtained.

Techniques Used: Activation Assay, Infection, Expressing, Isolation, Mouse Assay, Cell Culture, Staining, Flow Cytometry, Cytometry, Confocal Microscopy, Labeling

25) Product Images from "Interferon-? (IFN-?) suppresses HTLV-1 gene expression and cell cycling, while IFN-? combined with zidovudin induces p53 signaling and apoptosis in HTLV-1-infected cells"

Article Title: Interferon-? (IFN-?) suppresses HTLV-1 gene expression and cell cycling, while IFN-? combined with zidovudin induces p53 signaling and apoptosis in HTLV-1-infected cells

Journal: Retrovirology

doi: 10.1186/1742-4690-10-52

Effects of IFN-α treatment on HTLV-1 p19 release and viral transcription in various HTLV-1-infected cell lines. A . Expression of HTLV-1 mRNAs ( a ) and proteins ( b , c ) were evaluated by quantitative RT-PCR ( a ), immunoblotting ( b ), and flow cytometry ( c ), respectively, in HTLV-1-infected HUT102, ILT-Hod and ILT-#29 or uninfected Jurkat cell lines. a . The mRNA copy numbers measured by using pX or Gag primers were standardized to those for GAPDH and indicated as the means and standard deviations (SD) of duplicate samples. b . Cell lysates from indicated cell lines were subjected to an immunoblotting assay with antibodies to Tax (40 kDa) and α-Tubulin (50 kDa). The lysates in lanes 5 and 6 were prepared from ILT-Hod and ILT-#29 cells stimulated with PMA (50 ng/ml) overnight, respectively. c . Intracellular Tax proteins in permeabilized cells were stained with Alexa Fluor 488-labeled anti-Tax mAb (open histogram) and mouse IgG3 isotype control antibody (closed histogram). The inserted box indicates Gag expression in ILT-Hod and ILT-#29 cells stimulated with PMA (50 ng/ml) for 17h. B . HUT102 (top), ILT-Hod (middle) and ILT-#29 (bottom) cells were cultured for 3 days with or without three doses of IFN-α indicated. HTLV-1 p19 concentrations in the supernatants (left) and Gag mRNA levels were measured by ELISA and quantitative RT-PCR, respectively. Data are presented as the means and SD of duplicate samples. C . Frozen stored primary ATL cells were thawed and analyzed for intracellular Tax (top) or Gag (bottom) proteins by flow cytometry immediately (green line) or 24 h after culture with no (black line), 300 IU/ml (red line) or 3000 IU/ml (blue line) of IFN-α in the presence of IL-2 (30 IU/ml). The closed histogram represents samples stained with isotype controls. The mean fluorescence intensity (MFI) of each histogram was indicated in the bar graphs.
Figure Legend Snippet: Effects of IFN-α treatment on HTLV-1 p19 release and viral transcription in various HTLV-1-infected cell lines. A . Expression of HTLV-1 mRNAs ( a ) and proteins ( b , c ) were evaluated by quantitative RT-PCR ( a ), immunoblotting ( b ), and flow cytometry ( c ), respectively, in HTLV-1-infected HUT102, ILT-Hod and ILT-#29 or uninfected Jurkat cell lines. a . The mRNA copy numbers measured by using pX or Gag primers were standardized to those for GAPDH and indicated as the means and standard deviations (SD) of duplicate samples. b . Cell lysates from indicated cell lines were subjected to an immunoblotting assay with antibodies to Tax (40 kDa) and α-Tubulin (50 kDa). The lysates in lanes 5 and 6 were prepared from ILT-Hod and ILT-#29 cells stimulated with PMA (50 ng/ml) overnight, respectively. c . Intracellular Tax proteins in permeabilized cells were stained with Alexa Fluor 488-labeled anti-Tax mAb (open histogram) and mouse IgG3 isotype control antibody (closed histogram). The inserted box indicates Gag expression in ILT-Hod and ILT-#29 cells stimulated with PMA (50 ng/ml) for 17h. B . HUT102 (top), ILT-Hod (middle) and ILT-#29 (bottom) cells were cultured for 3 days with or without three doses of IFN-α indicated. HTLV-1 p19 concentrations in the supernatants (left) and Gag mRNA levels were measured by ELISA and quantitative RT-PCR, respectively. Data are presented as the means and SD of duplicate samples. C . Frozen stored primary ATL cells were thawed and analyzed for intracellular Tax (top) or Gag (bottom) proteins by flow cytometry immediately (green line) or 24 h after culture with no (black line), 300 IU/ml (red line) or 3000 IU/ml (blue line) of IFN-α in the presence of IL-2 (30 IU/ml). The closed histogram represents samples stained with isotype controls. The mean fluorescence intensity (MFI) of each histogram was indicated in the bar graphs.

Techniques Used: Infection, Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, Staining, Labeling, Cell Culture, Enzyme-linked Immunosorbent Assay, Fluorescence

26) Product Images from "Interferon-? (IFN-?) suppresses HTLV-1 gene expression and cell cycling, while IFN-? combined with zidovudin induces p53 signaling and apoptosis in HTLV-1-infected cells"

Article Title: Interferon-? (IFN-?) suppresses HTLV-1 gene expression and cell cycling, while IFN-? combined with zidovudin induces p53 signaling and apoptosis in HTLV-1-infected cells

Journal: Retrovirology

doi: 10.1186/1742-4690-10-52

Suppression of NF-κB activity by IFN-α in HTLV-1-infected cells. A . ILT-Hod and ILT-#29 cells that were infected with lentiviral vectors containing reporter gene for the NF-κB responsive element and the TK-promoter several weeks before, were treated with or without IFN-α (3000 IU/ml) and/or AZT (10 μM) for 4 days as indicated. Luciferase activities were measured, and relative NF-κB activities normalized to TK-promoter activities were indicated as means and SD of duplicate samples. *p
Figure Legend Snippet: Suppression of NF-κB activity by IFN-α in HTLV-1-infected cells. A . ILT-Hod and ILT-#29 cells that were infected with lentiviral vectors containing reporter gene for the NF-κB responsive element and the TK-promoter several weeks before, were treated with or without IFN-α (3000 IU/ml) and/or AZT (10 μM) for 4 days as indicated. Luciferase activities were measured, and relative NF-κB activities normalized to TK-promoter activities were indicated as means and SD of duplicate samples. *p

Techniques Used: Activity Assay, Infection, Luciferase

IFN-α suppressed Tax protein expression before an apparent reduction in HTLV-1 mRNA levels. A . The effects of IFN-α (3000 IU/ml) on intracellular Tax (top) and Gag (bottom) protein expression in ILT-Hod (left) and ILT-#29 (right) cells was evaluated by flow cytometry on days 1, 3, and 8 of culture. Cells stained with isotype antibodies served as negative controls. The values inside the dot plots represent percentages of viral protein-expressing cells, and the relative values in IFN-α-treated (closed bar) against untreated (open bar) samples are shown in the bar graph. The MFI value of the total cell population is indicated below the dot plots. B . Expression of HTLV-1 mRNA in the same cell samples prepared in A was evaluated by quantitative RT-PCR using pX (top) and Gag (bottom) primers. Results are standardized and presented as relative values of IFN-α-treated (closed bar) against untreated (open bar) samples. The means and SD of duplicate samples are indicated. *p
Figure Legend Snippet: IFN-α suppressed Tax protein expression before an apparent reduction in HTLV-1 mRNA levels. A . The effects of IFN-α (3000 IU/ml) on intracellular Tax (top) and Gag (bottom) protein expression in ILT-Hod (left) and ILT-#29 (right) cells was evaluated by flow cytometry on days 1, 3, and 8 of culture. Cells stained with isotype antibodies served as negative controls. The values inside the dot plots represent percentages of viral protein-expressing cells, and the relative values in IFN-α-treated (closed bar) against untreated (open bar) samples are shown in the bar graph. The MFI value of the total cell population is indicated below the dot plots. B . Expression of HTLV-1 mRNA in the same cell samples prepared in A was evaluated by quantitative RT-PCR using pX (top) and Gag (bottom) primers. Results are standardized and presented as relative values of IFN-α-treated (closed bar) against untreated (open bar) samples. The means and SD of duplicate samples are indicated. *p

Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Quantitative RT-PCR

Involvement of PKR in IFN-α-mediated reduction of Tax protein levels in HTLV-1-infected cells. A . ILT-Hod and ILT-#29 cells were incubated with or without the PKR-inhibitor (500 nM) or the negative control-inhibitor (500 nM) for the first 2 h, then further cultured for the next 24 h in the presence or absence of IFN-α (3000 IU/ml) as indicated. Flow cytometry was then performed following stained with anti-Tax (open histogram) and isotype control (closed histogram) antibodies. B . HTLV-1 pX mRNAs in the same samples prepared in A were quantified by RT-PCR using two different primer sets (RPX; black bar, and pX; gray bar), standardized to GAPDH mRNAs, and the relative values were indicated as the means and SD of duplicate samples. C . PKR mRNAs in ILT-Hod, ILT-#29, and HTLV-1-negative Jurkat and MOLT4 cells were quantified by RT-PCR, standardized to GAPDH mRNA and indicated as the means and SD of duplicate samples. D . PKR mRNAs in ILT-Hod and ILT-#29 cells were quantified 24 h after culture in the absence (open bar) or presence (closed bar) of IFN-α (3000 IU/ml), and the relative values are indicated as the means and SD of duplicate samples. *p
Figure Legend Snippet: Involvement of PKR in IFN-α-mediated reduction of Tax protein levels in HTLV-1-infected cells. A . ILT-Hod and ILT-#29 cells were incubated with or without the PKR-inhibitor (500 nM) or the negative control-inhibitor (500 nM) for the first 2 h, then further cultured for the next 24 h in the presence or absence of IFN-α (3000 IU/ml) as indicated. Flow cytometry was then performed following stained with anti-Tax (open histogram) and isotype control (closed histogram) antibodies. B . HTLV-1 pX mRNAs in the same samples prepared in A were quantified by RT-PCR using two different primer sets (RPX; black bar, and pX; gray bar), standardized to GAPDH mRNAs, and the relative values were indicated as the means and SD of duplicate samples. C . PKR mRNAs in ILT-Hod, ILT-#29, and HTLV-1-negative Jurkat and MOLT4 cells were quantified by RT-PCR, standardized to GAPDH mRNA and indicated as the means and SD of duplicate samples. D . PKR mRNAs in ILT-Hod and ILT-#29 cells were quantified 24 h after culture in the absence (open bar) or presence (closed bar) of IFN-α (3000 IU/ml), and the relative values are indicated as the means and SD of duplicate samples. *p

Techniques Used: Infection, Incubation, Negative Control, Cell Culture, Flow Cytometry, Cytometry, Staining, Reverse Transcription Polymerase Chain Reaction

Effects of IFN-α and AZT on HTLV-1 expression and cell growth of HTLV-1 infected cells. ILT-Hod and ILT-#29 cells (10 6 /ml) were cultured in the absence or presence of IFN-α (3000 IU/ml) and/or AZT (10 μM) as indicated, and HTLV-1 expression ( A ), cell growth ( B ), cell cycle ( C ), and Ki-67 expression ( D ) in the cells were evaluated. A . Expression of intracellular Tax protein 3 days after the initiation of culture was evaluated by flow cytometry following stained with anti-Tax (open histogram) and isotype control (closed histogram) antibodies. B . ILT-Hod and ILT-#29 cells were similarly treated with IFN-α and/or AZT, and maintained with addition of equal volumes of fresh medium without IFN-α or AZT on the day 1 and 3, then viable (closed bar) and non-viable (open bar) cell numbers in cultures were evaluated by trypan blue exclusion on the day 8. *p
Figure Legend Snippet: Effects of IFN-α and AZT on HTLV-1 expression and cell growth of HTLV-1 infected cells. ILT-Hod and ILT-#29 cells (10 6 /ml) were cultured in the absence or presence of IFN-α (3000 IU/ml) and/or AZT (10 μM) as indicated, and HTLV-1 expression ( A ), cell growth ( B ), cell cycle ( C ), and Ki-67 expression ( D ) in the cells were evaluated. A . Expression of intracellular Tax protein 3 days after the initiation of culture was evaluated by flow cytometry following stained with anti-Tax (open histogram) and isotype control (closed histogram) antibodies. B . ILT-Hod and ILT-#29 cells were similarly treated with IFN-α and/or AZT, and maintained with addition of equal volumes of fresh medium without IFN-α or AZT on the day 1 and 3, then viable (closed bar) and non-viable (open bar) cell numbers in cultures were evaluated by trypan blue exclusion on the day 8. *p

Techniques Used: Expressing, Infection, Cell Culture, Flow Cytometry, Cytometry, Staining

Induction of p53-signaling by IFN-α and AZT in HTLV-1-infected cells. A . Intracellular phosphorylated p53 levels in ILT-Hod and ILT-#29 cells were evaluated by flow cytomertry 3 and 4 days after incubation, respectively, in the absence (black line) or presence of IFN-α (3000 IU/ml) alone (blue line), or IFN-α/AZT (10 μM) (red line). The closed histograms indicate cells stained with control antibody. B . ILT-Hod and ILT-#29 were treated with IFN-α and/or AZT for 4 days and mRNA expression of BAX (closed bar) and p21 (open bar) was evaluated by quantitative RT-PCR. Results are standardized with the copy number of GAPDH mRNA, and the relative values are indicated as means and SD of duplicate samples. *p
Figure Legend Snippet: Induction of p53-signaling by IFN-α and AZT in HTLV-1-infected cells. A . Intracellular phosphorylated p53 levels in ILT-Hod and ILT-#29 cells were evaluated by flow cytomertry 3 and 4 days after incubation, respectively, in the absence (black line) or presence of IFN-α (3000 IU/ml) alone (blue line), or IFN-α/AZT (10 μM) (red line). The closed histograms indicate cells stained with control antibody. B . ILT-Hod and ILT-#29 were treated with IFN-α and/or AZT for 4 days and mRNA expression of BAX (closed bar) and p21 (open bar) was evaluated by quantitative RT-PCR. Results are standardized with the copy number of GAPDH mRNA, and the relative values are indicated as means and SD of duplicate samples. *p

Techniques Used: Infection, Flow Cytometry, Incubation, Staining, Expressing, Quantitative RT-PCR

Effects of IFN-α treatment on HTLV-1 p19 release and viral transcription in various HTLV-1-infected cell lines. A . Expression of HTLV-1 mRNAs ( a ) and proteins ( b , c ) were evaluated by quantitative RT-PCR ( a ), immunoblotting ( b ), and flow cytometry ( c ), respectively, in HTLV-1-infected HUT102, ILT-Hod and ILT-#29 or uninfected Jurkat cell lines. a . The mRNA copy numbers measured by using pX or Gag primers were standardized to those for GAPDH and indicated as the means and standard deviations (SD) of duplicate samples. b . Cell lysates from indicated cell lines were subjected to an immunoblotting assay with antibodies to Tax (40 kDa) and α-Tubulin (50 kDa). The lysates in lanes 5 and 6 were prepared from ILT-Hod and ILT-#29 cells stimulated with PMA (50 ng/ml) overnight, respectively. c . Intracellular Tax proteins in permeabilized cells were stained with Alexa Fluor 488-labeled anti-Tax mAb (open histogram) and mouse IgG3 isotype control antibody (closed histogram). The inserted box indicates Gag expression in ILT-Hod and ILT-#29 cells stimulated with PMA (50 ng/ml) for 17h. B . HUT102 (top), ILT-Hod (middle) and ILT-#29 (bottom) cells were cultured for 3 days with or without three doses of IFN-α indicated. HTLV-1 p19 concentrations in the supernatants (left) and Gag mRNA levels were measured by ELISA and quantitative RT-PCR, respectively. Data are presented as the means and SD of duplicate samples. C . Frozen stored primary ATL cells were thawed and analyzed for intracellular Tax (top) or Gag (bottom) proteins by flow cytometry immediately (green line) or 24 h after culture with no (black line), 300 IU/ml (red line) or 3000 IU/ml (blue line) of IFN-α in the presence of IL-2 (30 IU/ml). The closed histogram represents samples stained with isotype controls. The mean fluorescence intensity (MFI) of each histogram was indicated in the bar graphs.
Figure Legend Snippet: Effects of IFN-α treatment on HTLV-1 p19 release and viral transcription in various HTLV-1-infected cell lines. A . Expression of HTLV-1 mRNAs ( a ) and proteins ( b , c ) were evaluated by quantitative RT-PCR ( a ), immunoblotting ( b ), and flow cytometry ( c ), respectively, in HTLV-1-infected HUT102, ILT-Hod and ILT-#29 or uninfected Jurkat cell lines. a . The mRNA copy numbers measured by using pX or Gag primers were standardized to those for GAPDH and indicated as the means and standard deviations (SD) of duplicate samples. b . Cell lysates from indicated cell lines were subjected to an immunoblotting assay with antibodies to Tax (40 kDa) and α-Tubulin (50 kDa). The lysates in lanes 5 and 6 were prepared from ILT-Hod and ILT-#29 cells stimulated with PMA (50 ng/ml) overnight, respectively. c . Intracellular Tax proteins in permeabilized cells were stained with Alexa Fluor 488-labeled anti-Tax mAb (open histogram) and mouse IgG3 isotype control antibody (closed histogram). The inserted box indicates Gag expression in ILT-Hod and ILT-#29 cells stimulated with PMA (50 ng/ml) for 17h. B . HUT102 (top), ILT-Hod (middle) and ILT-#29 (bottom) cells were cultured for 3 days with or without three doses of IFN-α indicated. HTLV-1 p19 concentrations in the supernatants (left) and Gag mRNA levels were measured by ELISA and quantitative RT-PCR, respectively. Data are presented as the means and SD of duplicate samples. C . Frozen stored primary ATL cells were thawed and analyzed for intracellular Tax (top) or Gag (bottom) proteins by flow cytometry immediately (green line) or 24 h after culture with no (black line), 300 IU/ml (red line) or 3000 IU/ml (blue line) of IFN-α in the presence of IL-2 (30 IU/ml). The closed histogram represents samples stained with isotype controls. The mean fluorescence intensity (MFI) of each histogram was indicated in the bar graphs.

Techniques Used: Infection, Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, Staining, Labeling, Cell Culture, Enzyme-linked Immunosorbent Assay, Fluorescence

27) Product Images from "The Cytosolic Protein G0S2 Maintains Quiescence in Hematopoietic Stem Cells"

Article Title: The Cytosolic Protein G0S2 Maintains Quiescence in Hematopoietic Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0038280

Quiescent HSCs exhibit cytosolic sequestration of nucleolin. (A) Nucleolin colocalizes with the overexpressed G0S2 protein in LSK CD150 + CD48 − cells purified from mice transplanted with BM cells transduced with the MIGR1-G0S2 V5-tagged retrovirus. (B) Expression of nucleolin and Ki67 was determined in wild-type LS − K (proliferative progenitors), LSK CD150 + CD48 − cells purified from wild-type mice (dormant HSCs), and LSK CD150 + CD48 − cells purified from wild-type mice injected 6 days earlier with a single dose of 5-FU (proliferative HSCs). Images of DAPI, nucleolin, and Ki67 staining are shown for a representative cell. The data represent two independent experiments.
Figure Legend Snippet: Quiescent HSCs exhibit cytosolic sequestration of nucleolin. (A) Nucleolin colocalizes with the overexpressed G0S2 protein in LSK CD150 + CD48 − cells purified from mice transplanted with BM cells transduced with the MIGR1-G0S2 V5-tagged retrovirus. (B) Expression of nucleolin and Ki67 was determined in wild-type LS − K (proliferative progenitors), LSK CD150 + CD48 − cells purified from wild-type mice (dormant HSCs), and LSK CD150 + CD48 − cells purified from wild-type mice injected 6 days earlier with a single dose of 5-FU (proliferative HSCs). Images of DAPI, nucleolin, and Ki67 staining are shown for a representative cell. The data represent two independent experiments.

Techniques Used: Purification, Mouse Assay, Transduction, Expressing, Injection, Staining

G0S2 enhances the quiescence of hematopoietic stem cells. (A) To analyze the reconstitution potential of G0S2-overexpressing BM cells relative to wild-type BM cells, we performed a competitive transplantation with a mixture of BM cells transduced with the MIGR1 or MIGR1-G0S2 retrovirus ( n = 5). EGFP + CD45.1 + and EGFP + CD45.1 − cells were derived from MIGR1 and MIGR1-G0S2 BM cells, respectively. The data represent two independent experiments. (B) The role of G0S2 in HSC quiescence was examined using a gain-of-function model. Flow cytometric analyses of Ki67 and 7-AAD were performed in LSK CD150 + CD48 − cells purified from chimeric mice transplanted with cells containing the control or G0S2 retrovirus ( n = 3–4). Quiescent HSCs were defined as Ki67-negative cells (G 0 ) with a 2n DNA content. (C) Flow cytometric analyses of Ki67 and 7-AAD were performed in LSK CD150 + CD48 − cells purified from mice transplanted with G0S2-shRNA or the control retrovirus ( n = 4–5). *, P
Figure Legend Snippet: G0S2 enhances the quiescence of hematopoietic stem cells. (A) To analyze the reconstitution potential of G0S2-overexpressing BM cells relative to wild-type BM cells, we performed a competitive transplantation with a mixture of BM cells transduced with the MIGR1 or MIGR1-G0S2 retrovirus ( n = 5). EGFP + CD45.1 + and EGFP + CD45.1 − cells were derived from MIGR1 and MIGR1-G0S2 BM cells, respectively. The data represent two independent experiments. (B) The role of G0S2 in HSC quiescence was examined using a gain-of-function model. Flow cytometric analyses of Ki67 and 7-AAD were performed in LSK CD150 + CD48 − cells purified from chimeric mice transplanted with cells containing the control or G0S2 retrovirus ( n = 3–4). Quiescent HSCs were defined as Ki67-negative cells (G 0 ) with a 2n DNA content. (C) Flow cytometric analyses of Ki67 and 7-AAD were performed in LSK CD150 + CD48 − cells purified from mice transplanted with G0S2-shRNA or the control retrovirus ( n = 4–5). *, P

Techniques Used: Transplantation Assay, Transduction, Derivative Assay, Flow Cytometry, Purification, Mouse Assay, shRNA

G0S2 is expressed in dormant hematopoietic stem cells. (A) G0S2 transcripts were measured by quantitative real-time PCR in bone marrow hematopoietic stem and progenitor cells based on SLAM markers and mature myeloid and lymphoid cells purified from the spleen ( n = 3). Statistical significance is indicated between HSCs and progenitor cells (MPP, CMP, GMP, MEP). (B) Expression of G0S2 and cyclin E2 in BM cells isolated at different times after administration of a single dose of 5-FU in C57BL/6 mice. The relative expression levels of G0S2 and cyclin E2 are shown as percentages of basal levels ( n = 3–4). (C) Transcript levels of G0S2 and cyclin E2 were measured in LSK CD150 + CD48 − cells purified from untreated or 5-FU-treated (day 6) mice ( n = 3). The data represent the mean and standard deviation of each experiment. *, P
Figure Legend Snippet: G0S2 is expressed in dormant hematopoietic stem cells. (A) G0S2 transcripts were measured by quantitative real-time PCR in bone marrow hematopoietic stem and progenitor cells based on SLAM markers and mature myeloid and lymphoid cells purified from the spleen ( n = 3). Statistical significance is indicated between HSCs and progenitor cells (MPP, CMP, GMP, MEP). (B) Expression of G0S2 and cyclin E2 in BM cells isolated at different times after administration of a single dose of 5-FU in C57BL/6 mice. The relative expression levels of G0S2 and cyclin E2 are shown as percentages of basal levels ( n = 3–4). (C) Transcript levels of G0S2 and cyclin E2 were measured in LSK CD150 + CD48 − cells purified from untreated or 5-FU-treated (day 6) mice ( n = 3). The data represent the mean and standard deviation of each experiment. *, P

Techniques Used: Real-time Polymerase Chain Reaction, Purification, Expressing, Isolation, Mouse Assay, Standard Deviation

28) Product Images from "Essential complicity of perforin-granzyme and FAS-L mechanisms to achieve tumor rejection following treatment with anti-CD137 mAb"

Article Title: Essential complicity of perforin-granzyme and FAS-L mechanisms to achieve tumor rejection following treatment with anti-CD137 mAb

Journal: Journal for Immunotherapy of Cancer

doi: 10.1186/2051-1426-1-3

The cytotoxic mechanisms responsible for EG7 tumor rejection are present in TILs after treatment with anti-CD137 mAb. Mice were challenged s.c. with 5 × 10 5 EG7 cells and treated i.p. with anti-CD137 mAb on days 9 and 11. Two days later, tumors were removed and TILs were analyzed by flow cytometry for intracellular granzyme B ( A left) and percentage of H-2K b -SIINFEKL tetramer + cells ( A right), surface FasL ( B ), intracellular IFNγ ( C ) and surface CD107a ( D ) expression on gated CD8 + and CD4 + T lymphocytes. Histograms show CD8 + gated T cells that come from a representative experiment. Grey histograms represent isotype-matched control antibody stainings and open histograms protein-specific surface or intracellular stainings from representative cases. The insets in each histogram show the mean fluorescense intensity (MFI) after subtracting the background staining or the percentage of CD107a positive cells ( D ) for the indicated immunostainings on CD8 + and CD4 + T cells from individual tumors. The experiment was performed with 7-8 mice per group and mean±SEM is included in each histogram.
Figure Legend Snippet: The cytotoxic mechanisms responsible for EG7 tumor rejection are present in TILs after treatment with anti-CD137 mAb. Mice were challenged s.c. with 5 × 10 5 EG7 cells and treated i.p. with anti-CD137 mAb on days 9 and 11. Two days later, tumors were removed and TILs were analyzed by flow cytometry for intracellular granzyme B ( A left) and percentage of H-2K b -SIINFEKL tetramer + cells ( A right), surface FasL ( B ), intracellular IFNγ ( C ) and surface CD107a ( D ) expression on gated CD8 + and CD4 + T lymphocytes. Histograms show CD8 + gated T cells that come from a representative experiment. Grey histograms represent isotype-matched control antibody stainings and open histograms protein-specific surface or intracellular stainings from representative cases. The insets in each histogram show the mean fluorescense intensity (MFI) after subtracting the background staining or the percentage of CD107a positive cells ( D ) for the indicated immunostainings on CD8 + and CD4 + T cells from individual tumors. The experiment was performed with 7-8 mice per group and mean±SEM is included in each histogram.

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Staining

29) Product Images from "Clock gene Bmal1 is dispensable for intrinsic properties of murine hematopoietic stem cells"

Article Title: Clock gene Bmal1 is dispensable for intrinsic properties of murine hematopoietic stem cells

Journal: Journal of Negative Results in Biomedicine

doi: 10.1186/1477-5751-13-4

Cell cycling and differentiation of HSCs are normal in arrhythmic Bmal1 deficient mice. A, B) Normal frequency of HSCs in the BM of 8-10-week-old Bmal1 −/− mice. CD34 − KSL fractions were assessed by flow cytometry. A) Data shown are representative of CD34 − KSL cells at ZT5 and ZT17. B) The mean percentages ± SDs of CD34 − KSL cells at ZT5 (n = 4) and ZT17 (n = 3) of two independent experiments. C) Comparable frequency of quiescent cells in HSC populations. HSCs of Bmal1 +/+ and Bmal1 −/− mice were stained with Pyronin Y and analyzed by flow cytometry to give the mean percentages ± SDs of Pyronin Y − cells in the CD34 − KSL populations at ZT5 and ZT17 (n = 3) of two independent experiments. D) Normal EdU incorporation in Bmal1 −/− CD34 − KSL cells. EdU was administered orally to mice for 3 weeks, and EdU incorporation into HSCs was evaluated using a Click-iT EdU PB Flow Cytometry Assay Kit. Data shown are the mean percentages ± SDs of EdU + cells in HSC populations ( Bmal1 −/− mice; n = 6, Bmal1 −/− mice; n = 3). E) White blood cell differentiation in young (10-week-old) and aged (40-week-old) mice. Each stack in the bar represents a cell type percentage. Gr-1 + , granulocytes; Mac-1 + , macrophages; B220 + , B cells; CD4 + , CD4 + T cells; and CD8 + , CD8 + T cells (n = 6) of four independent experiments.
Figure Legend Snippet: Cell cycling and differentiation of HSCs are normal in arrhythmic Bmal1 deficient mice. A, B) Normal frequency of HSCs in the BM of 8-10-week-old Bmal1 −/− mice. CD34 − KSL fractions were assessed by flow cytometry. A) Data shown are representative of CD34 − KSL cells at ZT5 and ZT17. B) The mean percentages ± SDs of CD34 − KSL cells at ZT5 (n = 4) and ZT17 (n = 3) of two independent experiments. C) Comparable frequency of quiescent cells in HSC populations. HSCs of Bmal1 +/+ and Bmal1 −/− mice were stained with Pyronin Y and analyzed by flow cytometry to give the mean percentages ± SDs of Pyronin Y − cells in the CD34 − KSL populations at ZT5 and ZT17 (n = 3) of two independent experiments. D) Normal EdU incorporation in Bmal1 −/− CD34 − KSL cells. EdU was administered orally to mice for 3 weeks, and EdU incorporation into HSCs was evaluated using a Click-iT EdU PB Flow Cytometry Assay Kit. Data shown are the mean percentages ± SDs of EdU + cells in HSC populations ( Bmal1 −/− mice; n = 6, Bmal1 −/− mice; n = 3). E) White blood cell differentiation in young (10-week-old) and aged (40-week-old) mice. Each stack in the bar represents a cell type percentage. Gr-1 + , granulocytes; Mac-1 + , macrophages; B220 + , B cells; CD4 + , CD4 + T cells; and CD8 + , CD8 + T cells (n = 6) of four independent experiments.

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Staining, Cell Differentiation

30) Product Images from "CD4+ Natural Regulatory T Cells Prevent Experimental Cerebral Malaria via CTLA-4 When Expanded In Vivo"

Article Title: CD4+ Natural Regulatory T Cells Prevent Experimental Cerebral Malaria via CTLA-4 When Expanded In Vivo

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001221

Analysis of Treg cells expanded by IL-2Jc during ECM. A) Viable Foxp3 + CD4 + Treg cells (GFP pos ), and Foxp3 − CD4 + non-Treg cells (GFP neg ) were cell sorted from the spleens of naïve foxp3 gfp/gfp mice. 1×10 6 GFP pos cells or 5×10 6 GFP neg cells were adoptively transferred into mice, which were then infected with Pb A, and treated either with IL-2Jc or control saline. Four days later, numbers of splenic GFP + CD4 + T cells were enumerated. B) Naïve C57BL/6 mice, and those infected with Pb A, were treated with IL-2Jc or control saline. Four days later, spleens were isolated, Foxp3 + CD4 + T cells were enumerated by flow cytometry, and IL-10 and IFNγ production by these cells was assessed directly ex vivo by intracellular cytokine staining. C) Four days after infection with Pb A, splenic CD4 + Foxp3 + T cells from IL-2Jc treated and control saline treated mice we assessed for expression of CD25, Foxp3 and CTLA-4 by flow cytometry. Mann-Whitney: ***p
Figure Legend Snippet: Analysis of Treg cells expanded by IL-2Jc during ECM. A) Viable Foxp3 + CD4 + Treg cells (GFP pos ), and Foxp3 − CD4 + non-Treg cells (GFP neg ) were cell sorted from the spleens of naïve foxp3 gfp/gfp mice. 1×10 6 GFP pos cells or 5×10 6 GFP neg cells were adoptively transferred into mice, which were then infected with Pb A, and treated either with IL-2Jc or control saline. Four days later, numbers of splenic GFP + CD4 + T cells were enumerated. B) Naïve C57BL/6 mice, and those infected with Pb A, were treated with IL-2Jc or control saline. Four days later, spleens were isolated, Foxp3 + CD4 + T cells were enumerated by flow cytometry, and IL-10 and IFNγ production by these cells was assessed directly ex vivo by intracellular cytokine staining. C) Four days after infection with Pb A, splenic CD4 + Foxp3 + T cells from IL-2Jc treated and control saline treated mice we assessed for expression of CD25, Foxp3 and CTLA-4 by flow cytometry. Mann-Whitney: ***p

Techniques Used: Mouse Assay, Infection, Isolation, Flow Cytometry, Cytometry, Ex Vivo, Staining, Expressing, MANN-WHITNEY

IL-2Jc-mediated protection against ECM is dependent on Foxp3 + cells. C57BL/6 and DEREG mice (n = 5–6) were Pb A-infected and treated with IL-2Jc or saline on the same day. IL-2Jc-treated C57BL/6 and DEREG mice were i.p. treated on days 3, 5, 7 p.i. with 1µg of DT or saline. A) On day 4 p.i. blood CD4 + T cells were analysed by flow cytometry for the presence of GFP + Foxp3 + Treg cells. B) Mice (C57BL/6: upper graph)(DEREG: lower graph) were monitored for clinical symptoms from day 5 p.i. (120 hours p.i.). These data are representative of 2 independent experiments. C) C57BL/6 mice and DEREG mice (n = 5–7) were IL-2Jc or control saline treated on the day of infection with Pb A. All mice were DT treated, and on day 6 p.i. whole body parasite burdens were assessed. Mann-Whitney: **p
Figure Legend Snippet: IL-2Jc-mediated protection against ECM is dependent on Foxp3 + cells. C57BL/6 and DEREG mice (n = 5–6) were Pb A-infected and treated with IL-2Jc or saline on the same day. IL-2Jc-treated C57BL/6 and DEREG mice were i.p. treated on days 3, 5, 7 p.i. with 1µg of DT or saline. A) On day 4 p.i. blood CD4 + T cells were analysed by flow cytometry for the presence of GFP + Foxp3 + Treg cells. B) Mice (C57BL/6: upper graph)(DEREG: lower graph) were monitored for clinical symptoms from day 5 p.i. (120 hours p.i.). These data are representative of 2 independent experiments. C) C57BL/6 mice and DEREG mice (n = 5–7) were IL-2Jc or control saline treated on the day of infection with Pb A. All mice were DT treated, and on day 6 p.i. whole body parasite burdens were assessed. Mann-Whitney: **p

Techniques Used: Mouse Assay, Infection, Flow Cytometry, Cytometry, MANN-WHITNEY

IL-2Jc-mediated protection against ECM is dependent on CTLA-4 more than IL-10. C57BL/6 mice (n = 10–20 per group) were PbA -infected, and treated with IL-2Jc or control saline. On days 3 and 5 p.i. mice were i.p. treated with 0.5mg of anti-CTLA-4 mAb, anti-IL-10R mAb, or control rat IgG antibodies. Mice were monitored for A) survival, B) whole body parasite burden on day 6 p.i., with some groups also being analysed for C) splenic IFNγ + CD4 + T cell responses on day 4 p.i. Data in A) are pooled from 3 independent experiments, with statistical comparisons made relative to the “saline + Rat IgG” group. Log Rank tests performed in A), and Mann-Whitney tests in B) C) ***p
Figure Legend Snippet: IL-2Jc-mediated protection against ECM is dependent on CTLA-4 more than IL-10. C57BL/6 mice (n = 10–20 per group) were PbA -infected, and treated with IL-2Jc or control saline. On days 3 and 5 p.i. mice were i.p. treated with 0.5mg of anti-CTLA-4 mAb, anti-IL-10R mAb, or control rat IgG antibodies. Mice were monitored for A) survival, B) whole body parasite burden on day 6 p.i., with some groups also being analysed for C) splenic IFNγ + CD4 + T cell responses on day 4 p.i. Data in A) are pooled from 3 independent experiments, with statistical comparisons made relative to the “saline + Rat IgG” group. Log Rank tests performed in A), and Mann-Whitney tests in B) C) ***p

Techniques Used: Mouse Assay, Infection, MANN-WHITNEY

A proposed model for the roles of Treg cells in ECM. A). Following PbA infection of C57BL/6 mice, Treg cells suppress potent anti-parasitic T cells responses, but the PbA -specific T cell responses that do emerge promote parasite tissue sequestration, resulting in disease and death. However, if Treg cells can be expanded and activated sufficiently by early signalling via the high affinity IL-2 receptor, they can prevent pathogenic T cell responses mediating parasite tissue sequestration. B). The removal of CD25 hi Treg cells prior to PbA infection allows the generation of a potent anti-parasitic CD4 + T cell response, but also the re-emergence of CD25 hi Treg cells during the course of infection, resulting in an enhanced and qualitatively better anti-parasitic CD4 + T cells response that controls parasite growth and a mechanism to prevent parasite tissue sequestration. In contrast, if all Treg cells are depleted prior to infection, as is the case when DEREG mice are treated with DT, a balance between emerging anti-parasitic CD4 + T cell responses and expanding Treg cell responses is not achieved, and T cell-mediated immune pathology occurs. Conventional CD4 + (blue) and CD8 + (green) T cells, as well as CD25 hi (red) and CD25 lo or negative (purple) Treg cells are shown and disease outcome in different experimental conditions is shown.
Figure Legend Snippet: A proposed model for the roles of Treg cells in ECM. A). Following PbA infection of C57BL/6 mice, Treg cells suppress potent anti-parasitic T cells responses, but the PbA -specific T cell responses that do emerge promote parasite tissue sequestration, resulting in disease and death. However, if Treg cells can be expanded and activated sufficiently by early signalling via the high affinity IL-2 receptor, they can prevent pathogenic T cell responses mediating parasite tissue sequestration. B). The removal of CD25 hi Treg cells prior to PbA infection allows the generation of a potent anti-parasitic CD4 + T cell response, but also the re-emergence of CD25 hi Treg cells during the course of infection, resulting in an enhanced and qualitatively better anti-parasitic CD4 + T cells response that controls parasite growth and a mechanism to prevent parasite tissue sequestration. In contrast, if all Treg cells are depleted prior to infection, as is the case when DEREG mice are treated with DT, a balance between emerging anti-parasitic CD4 + T cell responses and expanding Treg cell responses is not achieved, and T cell-mediated immune pathology occurs. Conventional CD4 + (blue) and CD8 + (green) T cells, as well as CD25 hi (red) and CD25 lo or negative (purple) Treg cells are shown and disease outcome in different experimental conditions is shown.

Techniques Used: Infection, Mouse Assay

Total Foxp3 + cell depletion does not affect parasite burden or disease outcome in ECM. DEREG mice (n = 5) were treated with DT (1µg i.p.) or saline on the day prior to infection, and on days 2 and 4 after infection with Pb A. On day 4 p.i., A) Foxp3 + cell depletion and C) the magnitude of the IFNγ CD4 + T cell response was determined in the spleen by flow cytometry. B) Whole body parasite burdens were assessed on day 6 p.i.. These data are representative of 2 independent experiments. Mann-Whitney: *p
Figure Legend Snippet: Total Foxp3 + cell depletion does not affect parasite burden or disease outcome in ECM. DEREG mice (n = 5) were treated with DT (1µg i.p.) or saline on the day prior to infection, and on days 2 and 4 after infection with Pb A. On day 4 p.i., A) Foxp3 + cell depletion and C) the magnitude of the IFNγ CD4 + T cell response was determined in the spleen by flow cytometry. B) Whole body parasite burdens were assessed on day 6 p.i.. These data are representative of 2 independent experiments. Mann-Whitney: *p

Techniques Used: Mouse Assay, Infection, Flow Cytometry, Cytometry, MANN-WHITNEY

IL-2Jc-mediated protection is associated with impaired expansion of antigen-specific CD8 + T cells, reduced effector CD4 + T cell responses, and Treg cell expansion. A) C57BL/6 mice were adoptively transferred with 10,000 CD8 + CD45.1 + OTI T cells, and infected with SIINFEKL-expressing transgenic Pb TG. Control mice were infected with non-SIINFEKL-expressing Pb G parasites. Pb TG-infected mice were i.p. treated with IL-2Jc (day 0 or day 2), IL-2Sc (day 0), or control saline. On day 6 p.i., when control mice were displaying ECM symptoms, splenic CD8 + T cells (gated in FACS plots) were assessed for the presence of CD45.1 + SIINFEKL-specific T cells, and expression of the activation marker, GzmB. The graph indicates the number of CD45.1 + CD8 + SIINFEKL-specific T cells per spleen. Mann-Whitney * p
Figure Legend Snippet: IL-2Jc-mediated protection is associated with impaired expansion of antigen-specific CD8 + T cells, reduced effector CD4 + T cell responses, and Treg cell expansion. A) C57BL/6 mice were adoptively transferred with 10,000 CD8 + CD45.1 + OTI T cells, and infected with SIINFEKL-expressing transgenic Pb TG. Control mice were infected with non-SIINFEKL-expressing Pb G parasites. Pb TG-infected mice were i.p. treated with IL-2Jc (day 0 or day 2), IL-2Sc (day 0), or control saline. On day 6 p.i., when control mice were displaying ECM symptoms, splenic CD8 + T cells (gated in FACS plots) were assessed for the presence of CD45.1 + SIINFEKL-specific T cells, and expression of the activation marker, GzmB. The graph indicates the number of CD45.1 + CD8 + SIINFEKL-specific T cells per spleen. Mann-Whitney * p

Techniques Used: Mouse Assay, Infection, Expressing, Transgenic Assay, FACS, Activation Assay, Marker, MANN-WHITNEY

31) Product Images from "What Makes Umbilical Cord Tissue-Derived Mesenchymal Stromal Cells Superior Immunomodulators When Compared to Bone Marrow Derived Mesenchymal Stromal Cells?"

Article Title: What Makes Umbilical Cord Tissue-Derived Mesenchymal Stromal Cells Superior Immunomodulators When Compared to Bone Marrow Derived Mesenchymal Stromal Cells?

Journal: Stem Cells International

doi: 10.1155/2015/583984

Flow cytometry surface expression studies of CD200, CD273, and CD274 in UCX and BM-MSCs. (a) Representative histograms of staining for the three surface proteins in UCX (grey lines) and BM-MSCs (black lines) and respective isotype controls. (b) Graphical representation of the percentage of cells positive for each surface protein from 3 different samples of each cell type (between passages 5 and 7) and presented as mean ± s.e.m. A higher percentage of UCX cells constitutively express CD200, CD73, and CD274 when compared to BM-MSCs.
Figure Legend Snippet: Flow cytometry surface expression studies of CD200, CD273, and CD274 in UCX and BM-MSCs. (a) Representative histograms of staining for the three surface proteins in UCX (grey lines) and BM-MSCs (black lines) and respective isotype controls. (b) Graphical representation of the percentage of cells positive for each surface protein from 3 different samples of each cell type (between passages 5 and 7) and presented as mean ± s.e.m. A higher percentage of UCX cells constitutively express CD200, CD73, and CD274 when compared to BM-MSCs.

Techniques Used: Flow Cytometry, Cytometry, Expressing, Staining

32) Product Images from "Loss of signaling via Gα13 in germinal center B cell-derived lymphoma"

Article Title: Loss of signaling via Gα13 in germinal center B cell-derived lymphoma

Journal: Nature

doi: 10.1038/nature13765

Frequency of mutations in GNA13, S1PR2 and P2RY8 in aggressive lymphoma (a-b) Summary of overall mutation frequencies (a) and allelic frequencies (b) of non-synonymous coding mutations in S1PR2 , GNA13 and P2RY8 in GCB-DLBCL, BL or ABC-DLBCL cases shown in Supplementary Table 2 . Unmutated indicates no coding region mutations in the genes shown. Since the sequencing was performed on genomic DNA the data may underestimate the frequency of biallelic cases as some disruptive mutations may occur in non-coding regulatory elements.
Figure Legend Snippet: Frequency of mutations in GNA13, S1PR2 and P2RY8 in aggressive lymphoma (a-b) Summary of overall mutation frequencies (a) and allelic frequencies (b) of non-synonymous coding mutations in S1PR2 , GNA13 and P2RY8 in GCB-DLBCL, BL or ABC-DLBCL cases shown in Supplementary Table 2 . Unmutated indicates no coding region mutations in the genes shown. Since the sequencing was performed on genomic DNA the data may underestimate the frequency of biallelic cases as some disruptive mutations may occur in non-coding regulatory elements.

Techniques Used: Mutagenesis, Sequencing

P2RY8, mutated in GCB-DLBCL and BL, suppresses GC B cell growth and promotes B cell confinement via Gα 13 (a) Schematic of P2RY8 with locations of mutated residues in GCB-DLBCL and BL. Residues are marked as for S1PR2 in Figure 1a . (b) Phylogenetic tree of P2RY8 across species. (c) Quantitative PCR of S1PR1 , S1PR2 and P2RY8 in FACS-sorted human tonsillar Fo and GC B cells. Data in c are from 5 donors. (d, e) Fold change in frequency of Thy1.1 reporter + cells in GC relative to Fo B cells of PPs from BM chimeras reconstituted with S1pr2 KO BM (d) or Gna13 KO ( f/f mb1-cre ) BM (e) transduced with retrovirus expressing P2RY8, or with S1PR2, GNA13 or R147C mutant S1PR2 (control). Data in d are pooled from 2 independent experiments (S1PR2, n=4; Control, n=8; P2RY8, n=8). Data in e are from one experiment (n=4 in each group). (f, g) Immunohistochemical analysis of splenic sections from SRBC-immunized mice given Ig-transgenic (f) or Gna13 WT or KO (g) B cells transduced with retroviral vector encoding Thy1.1 alone (vector) or P2RY8 and Thy1.1, assessed 24 h after cell transfer. Scale bar is 200 μm in f and g. Data in f are representative of 3 and in g of 2 independent experiments. * P
Figure Legend Snippet: P2RY8, mutated in GCB-DLBCL and BL, suppresses GC B cell growth and promotes B cell confinement via Gα 13 (a) Schematic of P2RY8 with locations of mutated residues in GCB-DLBCL and BL. Residues are marked as for S1PR2 in Figure 1a . (b) Phylogenetic tree of P2RY8 across species. (c) Quantitative PCR of S1PR1 , S1PR2 and P2RY8 in FACS-sorted human tonsillar Fo and GC B cells. Data in c are from 5 donors. (d, e) Fold change in frequency of Thy1.1 reporter + cells in GC relative to Fo B cells of PPs from BM chimeras reconstituted with S1pr2 KO BM (d) or Gna13 KO ( f/f mb1-cre ) BM (e) transduced with retrovirus expressing P2RY8, or with S1PR2, GNA13 or R147C mutant S1PR2 (control). Data in d are pooled from 2 independent experiments (S1PR2, n=4; Control, n=8; P2RY8, n=8). Data in e are from one experiment (n=4 in each group). (f, g) Immunohistochemical analysis of splenic sections from SRBC-immunized mice given Ig-transgenic (f) or Gna13 WT or KO (g) B cells transduced with retroviral vector encoding Thy1.1 alone (vector) or P2RY8 and Thy1.1, assessed 24 h after cell transfer. Scale bar is 200 μm in f and g. Data in f are representative of 3 and in g of 2 independent experiments. * P

Techniques Used: Real-time Polymerase Chain Reaction, FACS, Transduction, Expressing, Mutagenesis, Immunohistochemistry, Mouse Assay, Transgenic Assay, Plasmid Preparation

Defective regulation of pAkt and cell migration in human GCB DLBCL cell lines harboring mutations in the S1PR2 signaling pathway (a) Frequency of non-synonymous coding mutations in S1PR2 , GNA13 and ARHGEF1 in GCB-DLBCL lines, and fraction that were mono- or bi-allelic, summarized from Supplementary Table 3 . Unmutated indicates no coding region mutations in the genes shown. (b, c) Intracellular FACS (b) or Western blot (c) for pAkt in human GCB DLBCL cell lines that are WT or mutant for S1PR2 , GNA13 or ARHGEF1 as indicated and that were stimulated with CXCL12 (100 ng/ml) in the presence or absence of S1P (10 nM) for 5 minutes. pAkt staining of cells treated with wortmannin (200 nM) for 5 minutes are shown in gray as a staining control for each cell line. (d) Transwell migration of GNA13 WT (Ly7, Ly8, NUDUL1) or mutant (DOHH2) cell lines to CXCL12 (100 ng/ml) in the presence or absence of S1P (10 nM). (e, f) Intracellular FACS for pAkt of the GNA13 mutant cell lines Karpas422 (d) or DOHH (e) transduced with retrovirus expressing the reporter alone (vector) or GNA13 in the presence or absence of S1P (10 nM) or wortmannin (200 nM; staining control). (g) Intracellular FACS for pAkt in the ARHGEF1 mutant cell line Ly19 transduced with retrovirus expressing reporter alone (vector) or ARHGEF1 that were treated as in b or with the PI3K inhibitor GS-1101 (2 uM; staining control). (h) Quantitative PCR analysis of S1PR2 transcript abundance in human GCB-DLBCL cell lines relative to GAPDH . (i) Intracellular FACS for pAkt in NUDUL1 cells transduced with retrovirus expressing reporter alone (vector), S1PR2, GNA13 or ARHGEF1, treated as in d. Data in b and d are representative of at least 3 independent experiments. Pooled data from at least 3 independent experiments is shown in b, e, f, g and i. Data in b is one experiment representative of 2. ** P
Figure Legend Snippet: Defective regulation of pAkt and cell migration in human GCB DLBCL cell lines harboring mutations in the S1PR2 signaling pathway (a) Frequency of non-synonymous coding mutations in S1PR2 , GNA13 and ARHGEF1 in GCB-DLBCL lines, and fraction that were mono- or bi-allelic, summarized from Supplementary Table 3 . Unmutated indicates no coding region mutations in the genes shown. (b, c) Intracellular FACS (b) or Western blot (c) for pAkt in human GCB DLBCL cell lines that are WT or mutant for S1PR2 , GNA13 or ARHGEF1 as indicated and that were stimulated with CXCL12 (100 ng/ml) in the presence or absence of S1P (10 nM) for 5 minutes. pAkt staining of cells treated with wortmannin (200 nM) for 5 minutes are shown in gray as a staining control for each cell line. (d) Transwell migration of GNA13 WT (Ly7, Ly8, NUDUL1) or mutant (DOHH2) cell lines to CXCL12 (100 ng/ml) in the presence or absence of S1P (10 nM). (e, f) Intracellular FACS for pAkt of the GNA13 mutant cell lines Karpas422 (d) or DOHH (e) transduced with retrovirus expressing the reporter alone (vector) or GNA13 in the presence or absence of S1P (10 nM) or wortmannin (200 nM; staining control). (g) Intracellular FACS for pAkt in the ARHGEF1 mutant cell line Ly19 transduced with retrovirus expressing reporter alone (vector) or ARHGEF1 that were treated as in b or with the PI3K inhibitor GS-1101 (2 uM; staining control). (h) Quantitative PCR analysis of S1PR2 transcript abundance in human GCB-DLBCL cell lines relative to GAPDH . (i) Intracellular FACS for pAkt in NUDUL1 cells transduced with retrovirus expressing reporter alone (vector), S1PR2, GNA13 or ARHGEF1, treated as in d. Data in b and d are representative of at least 3 independent experiments. Pooled data from at least 3 independent experiments is shown in b, e, f, g and i. Data in b is one experiment representative of 2. ** P

Techniques Used: Migration, FACS, Western Blot, Mutagenesis, Staining, Transduction, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction

Human P2RY8 suppresses GC B cell growth and promotes B cell confinement to the GC in mice (a, b) P2RY8 mutations arising in GCB-DLBCL and BL disrupt receptor expression. Flag-tagged versions of six point mutant and the wild-type receptor were expressed in WEH231 B cells and surface expression examined by FLAG flow cytometry (a). The transduction efficiency of each construct was confirmed to be similar based on IRES-Thy 1.1 reporter expression (b). (c, d) Fold change in Thy1.1 reporter + GC relative to Fo B cells from mLN of chimeras described in Fig. 4d-e . (e-g) Immunohistochemical analysis of splenic sections from SRBC-immunized mice given Ig-transgenic (e), Gpr183 +/- (f) or Gna13 WT or KO (g) B cells transduced as in Fig. 4f and g and assessed 24 h after cell transfer. Data in e and g are additional examples of the experiments shown in Fig. 4f and g , respectively. Data in f are representative of 4 independent experiments. Scale bar is 200 μm in e-g. * P
Figure Legend Snippet: Human P2RY8 suppresses GC B cell growth and promotes B cell confinement to the GC in mice (a, b) P2RY8 mutations arising in GCB-DLBCL and BL disrupt receptor expression. Flag-tagged versions of six point mutant and the wild-type receptor were expressed in WEH231 B cells and surface expression examined by FLAG flow cytometry (a). The transduction efficiency of each construct was confirmed to be similar based on IRES-Thy 1.1 reporter expression (b). (c, d) Fold change in Thy1.1 reporter + GC relative to Fo B cells from mLN of chimeras described in Fig. 4d-e . (e-g) Immunohistochemical analysis of splenic sections from SRBC-immunized mice given Ig-transgenic (e), Gpr183 +/- (f) or Gna13 WT or KO (g) B cells transduced as in Fig. 4f and g and assessed 24 h after cell transfer. Data in e and g are additional examples of the experiments shown in Fig. 4f and g , respectively. Data in f are representative of 4 independent experiments. Scale bar is 200 μm in e-g. * P

Techniques Used: Mouse Assay, Expressing, Mutagenesis, Flow Cytometry, Cytometry, Transduction, Construct, Immunohistochemistry, Transgenic Assay

33) Product Images from "Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation"

Article Title: Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2010.01012.x

Measurement of HAb18G/CD147 expression on resting and activated CD 24+ and CD 8+ T cell subsets. Purified resting T cells and activated T cells were stained with PE- conjugated anti-CD147, FITC-conjugated anti-CD4 or CD8, and were analysed by flow cytometry. (A) Histogram shows expression of CD147 on resting and activated CD 24+ T cells. (B) Histogram shows expression of CD147 on resting and activated CD 8+ T cells. These data are representative of a minimum of three separate experiments.
Figure Legend Snippet: Measurement of HAb18G/CD147 expression on resting and activated CD 24+ and CD 8+ T cell subsets. Purified resting T cells and activated T cells were stained with PE- conjugated anti-CD147, FITC-conjugated anti-CD4 or CD8, and were analysed by flow cytometry. (A) Histogram shows expression of CD147 on resting and activated CD 24+ T cells. (B) Histogram shows expression of CD147 on resting and activated CD 8+ T cells. These data are representative of a minimum of three separate experiments.

Techniques Used: Expressing, Purification, Staining, Flow Cytometry, Cytometry

34) Product Images from "Novel Fat Depot-Specific Mechanisms Underlie Resistance to Visceral Obesity and Inflammation in 11?-Hydroxysteroid Dehydrogenase Type 1-Deficient Mice"

Article Title: Novel Fat Depot-Specific Mechanisms Underlie Resistance to Visceral Obesity and Inflammation in 11?-Hydroxysteroid Dehydrogenase Type 1-Deficient Mice

Journal: Diabetes

doi: 10.2337/db10-0830

T-cell levels in adipose tissues of C57BL/6J and 11β-HSD1 −/− mice fed HF diet for 4 weeks. A : Anti-CD3 staining in mesenteric adipose sections from C57BL/6J (B6) and 11β-HSD1 −/− mice (KO) fed control or HF diet (B6HF, KOHF) (representative of n = 5). Note fat cell expansion causes the appearance of lower CD3 + cells/area, but there is actually an increase per depot as shown in B . FACS quantification of T-cell numbers in mesenteric ( B ) adipose SVC, and ( C ) adipose lymph nodes from C57BL/6J mice fed control (■) or HF (▨) diet and 11β-HSD1 −/− mice fed control (□) or HF (▤) diet. CD8+ cytotoxic T-cells are shown on the left , and CD3 + CD8 − (a surrogate for CD4 + T-helper cells) FACS data are shown on the right ; n = 4, with adipose pooled from two mice per condition. Effects of diet are shown as significant: † P
Figure Legend Snippet: T-cell levels in adipose tissues of C57BL/6J and 11β-HSD1 −/− mice fed HF diet for 4 weeks. A : Anti-CD3 staining in mesenteric adipose sections from C57BL/6J (B6) and 11β-HSD1 −/− mice (KO) fed control or HF diet (B6HF, KOHF) (representative of n = 5). Note fat cell expansion causes the appearance of lower CD3 + cells/area, but there is actually an increase per depot as shown in B . FACS quantification of T-cell numbers in mesenteric ( B ) adipose SVC, and ( C ) adipose lymph nodes from C57BL/6J mice fed control (■) or HF (▨) diet and 11β-HSD1 −/− mice fed control (□) or HF (▤) diet. CD8+ cytotoxic T-cells are shown on the left , and CD3 + CD8 − (a surrogate for CD4 + T-helper cells) FACS data are shown on the right ; n = 4, with adipose pooled from two mice per condition. Effects of diet are shown as significant: † P

Techniques Used: Mouse Assay, Staining, FACS

35) Product Images from "Type I interferon is selectively required by dendritic cells for immune rejection of tumors"

Article Title: Type I interferon is selectively required by dendritic cells for immune rejection of tumors

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20101158

NK cell depletion does not abrogate IFN-α/β–dependent rejection of immunogenic sarcomas. (A and B) C57BL/6 WT, Rag2 −/− , and Ifnar1 −/− mice and WT mice treated with either PBS or anti-NK1.1 PK136 mAb were injected s.c. (10 6 cells/mouse) with 1969 (A) or 7835 (B) unedited MCA sarcoma cells, and growth was monitored over time. Data are presented as mean tumor diameter ± SEM of 4–13 (untreated) or 8 (treated) mice per group from at least two independent experiments. Error bars for Ifnar1 −/− mice reflect progressive growth of 1969 and 7835 tumors in 6/9 mice. (C) WT C57BL/6 mice were treated with either PBS or PK136 mAb, and splenocytes were harvested 2 d later and analyzed by flow cytometry using the NK cell markers DX5 and NKp46. Splenocytes were gated on CD3 − cells, and the percentages of DX5 + NKp46 + cells are indicated. Similar results were found when harvested at day 6 (not depicted). (D) WT C57BL/6 mice were treated with PBS or PK136 followed by i.p. injection of 300 µg polyI:C 4 d later. After 24 h, splenocytes were harvested and used as effectors in a standard 4-h cytotoxicity assay with NK-sensitive YAC-1 targets. Specific lysis (in percentage ± SEM) at the indicated effector/target (E:T) ratios is shown for four mice/group assayed in duplicate from two independent experiments. (E) WT C57BL/6 mice were treated with PBS, PK136, or a mixture of anti-CD4 (GK1.5) and anti-CD8 (YTS-169.4) mAbs and injected s.c. with 10 5 RMA-S cells, and tumor growth was monitored over time. Mean tumor diameter ± SEM for three mice/group is shown, and data are representative of two independent experiments.
Figure Legend Snippet: NK cell depletion does not abrogate IFN-α/β–dependent rejection of immunogenic sarcomas. (A and B) C57BL/6 WT, Rag2 −/− , and Ifnar1 −/− mice and WT mice treated with either PBS or anti-NK1.1 PK136 mAb were injected s.c. (10 6 cells/mouse) with 1969 (A) or 7835 (B) unedited MCA sarcoma cells, and growth was monitored over time. Data are presented as mean tumor diameter ± SEM of 4–13 (untreated) or 8 (treated) mice per group from at least two independent experiments. Error bars for Ifnar1 −/− mice reflect progressive growth of 1969 and 7835 tumors in 6/9 mice. (C) WT C57BL/6 mice were treated with either PBS or PK136 mAb, and splenocytes were harvested 2 d later and analyzed by flow cytometry using the NK cell markers DX5 and NKp46. Splenocytes were gated on CD3 − cells, and the percentages of DX5 + NKp46 + cells are indicated. Similar results were found when harvested at day 6 (not depicted). (D) WT C57BL/6 mice were treated with PBS or PK136 followed by i.p. injection of 300 µg polyI:C 4 d later. After 24 h, splenocytes were harvested and used as effectors in a standard 4-h cytotoxicity assay with NK-sensitive YAC-1 targets. Specific lysis (in percentage ± SEM) at the indicated effector/target (E:T) ratios is shown for four mice/group assayed in duplicate from two independent experiments. (E) WT C57BL/6 mice were treated with PBS, PK136, or a mixture of anti-CD4 (GK1.5) and anti-CD8 (YTS-169.4) mAbs and injected s.c. with 10 5 RMA-S cells, and tumor growth was monitored over time. Mean tumor diameter ± SEM for three mice/group is shown, and data are representative of two independent experiments.

Techniques Used: Mouse Assay, Injection, Flow Cytometry, Cytometry, Cytotoxicity Assay, Lysis

36) Product Images from "TIM-3 Expression Characterizes Regulatory T Cells in Tumor Tissues and Is Associated with Lung Cancer Progression"

Article Title: TIM-3 Expression Characterizes Regulatory T Cells in Tumor Tissues and Is Associated with Lung Cancer Progression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0030676

TIM-3 expression on CD4 + TILs and Treg. TILs were harvested from lung cancer tissue. Cells were then stained for CD4, TIM-3, and FOXP3 (A) or CD4, PD-1, and FOXP3 (B). Lymphocytes were gated for further analysis of CD4 and CD8 T cells. The percentage of each population within CD4 + T cell compartment was indicated. Data shown are representative of five independent experiments.
Figure Legend Snippet: TIM-3 expression on CD4 + TILs and Treg. TILs were harvested from lung cancer tissue. Cells were then stained for CD4, TIM-3, and FOXP3 (A) or CD4, PD-1, and FOXP3 (B). Lymphocytes were gated for further analysis of CD4 and CD8 T cells. The percentage of each population within CD4 + T cell compartment was indicated. Data shown are representative of five independent experiments.

Techniques Used: Expressing, Staining

Expression of Tim-3 on TILs in mouse model of transplantable tumor. 6–8-week-old C57BL/6 mice were inoculated with 2×10 5 B16F0 cells i.d., tumor samples, spleens, and lymph nodes were removed when tumor sizes reached around 15 mm in diameters on day 20. TILs, splenocytes, and lymph node cells were isolated for analysis by flow cytometry. Tim-3 expression on mouse CD4 + Foxp3 + or CD4 + Foxp3 − T cells is shown. Data shown are representative of five independent experiments.
Figure Legend Snippet: Expression of Tim-3 on TILs in mouse model of transplantable tumor. 6–8-week-old C57BL/6 mice were inoculated with 2×10 5 B16F0 cells i.d., tumor samples, spleens, and lymph nodes were removed when tumor sizes reached around 15 mm in diameters on day 20. TILs, splenocytes, and lymph node cells were isolated for analysis by flow cytometry. Tim-3 expression on mouse CD4 + Foxp3 + or CD4 + Foxp3 − T cells is shown. Data shown are representative of five independent experiments.

Techniques Used: Expressing, Mouse Assay, Isolation, Flow Cytometry, Cytometry

TIM-3 and PD-1 were up-regulated upon T cell activation. (A and B). Human naïve T cells were purified from PBMCs of health donors. Cells were then stimulated with plate-bound anti-CD3 plus anti-CD28 mAb. 24, 48 and 72 hours later, cells were collected and stained for CD4, CD8, TIM-3 and PD-1. The expression of PD-1 and TIM-3 was analyzed by flow cytometry on gated CD4 + (A) or CD8 + (B) T cells. C. CD4 + CD25 high T cells were isolated from peripheral blood by FACS. These cells were then stimulated with anti-CD3 and anti-CD28 in the presence of IL-2 (200 U/mL). 24, 48 and 72 hours later, cells were harvested and stained for CD4, TIM-3 and FOXP3. The expression of TIM-3 on CD4 + FOXP3 + T cells is shown. Data are representative of three independent experiments.
Figure Legend Snippet: TIM-3 and PD-1 were up-regulated upon T cell activation. (A and B). Human naïve T cells were purified from PBMCs of health donors. Cells were then stimulated with plate-bound anti-CD3 plus anti-CD28 mAb. 24, 48 and 72 hours later, cells were collected and stained for CD4, CD8, TIM-3 and PD-1. The expression of PD-1 and TIM-3 was analyzed by flow cytometry on gated CD4 + (A) or CD8 + (B) T cells. C. CD4 + CD25 high T cells were isolated from peripheral blood by FACS. These cells were then stimulated with anti-CD3 and anti-CD28 in the presence of IL-2 (200 U/mL). 24, 48 and 72 hours later, cells were harvested and stained for CD4, TIM-3 and FOXP3. The expression of TIM-3 on CD4 + FOXP3 + T cells is shown. Data are representative of three independent experiments.

Techniques Used: Activation Assay, Purification, Staining, Expressing, Flow Cytometry, Cytometry, Isolation, FACS

37) Product Images from "Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection"

Article Title: Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004849

Clonal expansions of CD8+ T cells specific for the immunodominant antigen TB10.4. ( a ) Clonality of CD8+ TCR sequences obtained from infected lung granulomas (n = 6) compared to splenocytes from uninfected mice (n = 9); p = 0.0004 by Mann-Whitney. ( b,c ) Frequency of TCR Vβ families ( b ) and CDR3βs ( c ) of TB10.4 4-11 -specific CD8 + T cells from the lungs of C57BL/6J mice infected with M . tuberculosis for 9 weeks. Data were obtained by Next-generation sequencing of tetramer-purified T cells from 6 individual mice. ( d ) Frequency of TCR Vβ4 (TRBV2), Vβ5 (TRBV12), Vβ7 (TRBV29), Vβ10 (TRBV4) and Vβ11 (TRBV16) families of TB10.4 4-11 -tetramer + CD8 + T (red) or TB10.4 4-11 -tetramer - CD8 + T (blue) cells from the pulmonary LN of C57BL/6J mice infected with M . tuberculosis at 21 days post infection. ( e ) Frequency of TCR Vβ4 (TRBV2), Vβ7 (TRBV29) and Vβ10 (TRBV4) families of CD8 + T cells from the pulmonary LN or lung of C57BL/6J mice infected with M . tuberculosis at 21 days (left panel), 28 days (middle panel) or > 25 weeks (right panel) post infection. Dotted lines connect data from individual mice, some of which are labeled (A through I) to highlight mice with biased Vβ family usage. Data were obtained by flow cytometric analysis of TB10.4 4-11 -tetramer + CD8 + T cells (Tet+) or TB10.4 4-11 -tetramer - CD8 + T cells (Tet-) from three independent experiments, each with 4–10 mice per group.
Figure Legend Snippet: Clonal expansions of CD8+ T cells specific for the immunodominant antigen TB10.4. ( a ) Clonality of CD8+ TCR sequences obtained from infected lung granulomas (n = 6) compared to splenocytes from uninfected mice (n = 9); p = 0.0004 by Mann-Whitney. ( b,c ) Frequency of TCR Vβ families ( b ) and CDR3βs ( c ) of TB10.4 4-11 -specific CD8 + T cells from the lungs of C57BL/6J mice infected with M . tuberculosis for 9 weeks. Data were obtained by Next-generation sequencing of tetramer-purified T cells from 6 individual mice. ( d ) Frequency of TCR Vβ4 (TRBV2), Vβ5 (TRBV12), Vβ7 (TRBV29), Vβ10 (TRBV4) and Vβ11 (TRBV16) families of TB10.4 4-11 -tetramer + CD8 + T (red) or TB10.4 4-11 -tetramer - CD8 + T (blue) cells from the pulmonary LN of C57BL/6J mice infected with M . tuberculosis at 21 days post infection. ( e ) Frequency of TCR Vβ4 (TRBV2), Vβ7 (TRBV29) and Vβ10 (TRBV4) families of CD8 + T cells from the pulmonary LN or lung of C57BL/6J mice infected with M . tuberculosis at 21 days (left panel), 28 days (middle panel) or > 25 weeks (right panel) post infection. Dotted lines connect data from individual mice, some of which are labeled (A through I) to highlight mice with biased Vβ family usage. Data were obtained by flow cytometric analysis of TB10.4 4-11 -tetramer + CD8 + T cells (Tet+) or TB10.4 4-11 -tetramer - CD8 + T cells (Tet-) from three independent experiments, each with 4–10 mice per group.

Techniques Used: Infection, Mouse Assay, MANN-WHITNEY, Next-Generation Sequencing, Purification, Labeling, Flow Cytometry

38) Product Images from "Dysregulation of Th17 Cells during the Early Post-Transplant Period in Patients under Calcineurin Inhibitor Based Immunosuppression"

Article Title: Dysregulation of Th17 Cells during the Early Post-Transplant Period in Patients under Calcineurin Inhibitor Based Immunosuppression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0042011

Distribution of T naïve , T CM , T EM subpopulations and IL-17 + /T naïve , IL-17 + /T EM and IL-17 + /T CM , subpopulations of CD4 + T lymphocytes at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of GolgiStop.CD4 + lymphocytes were stained with mAbs to CD45RA and CCR7, which identified three subsets. In addition, analysis of IL-17 in CD4 + T cell subsets by intracellular flow cytometry was done. After surface staining with CD45 and CCR7 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with IL-17 mAbs. (A) T naïve /CD4 + T (CD45RA + CCR7 + /CD4 + Tcells), (B) IL-17 + /T naïve , (C) T CM /CD4 + T (CD45RA − CCR7 + /CD4 + Tcells), (D) IL-17 + /T CM + , (E) T EM /CD4 + T (CD45RA − CCR7 − /CD4 + Tcells), (F) IL-17 + /T EM + . Bars show the means. * P
Figure Legend Snippet: Distribution of T naïve , T CM , T EM subpopulations and IL-17 + /T naïve , IL-17 + /T EM and IL-17 + /T CM , subpopulations of CD4 + T lymphocytes at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of GolgiStop.CD4 + lymphocytes were stained with mAbs to CD45RA and CCR7, which identified three subsets. In addition, analysis of IL-17 in CD4 + T cell subsets by intracellular flow cytometry was done. After surface staining with CD45 and CCR7 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with IL-17 mAbs. (A) T naïve /CD4 + T (CD45RA + CCR7 + /CD4 + Tcells), (B) IL-17 + /T naïve , (C) T CM /CD4 + T (CD45RA − CCR7 + /CD4 + Tcells), (D) IL-17 + /T CM + , (E) T EM /CD4 + T (CD45RA − CCR7 − /CD4 + Tcells), (F) IL-17 + /T EM + . Bars show the means. * P

Techniques Used: Transplantation Assay, Ex Vivo, Staining, Flow Cytometry, Cytometry

Flow cytometric analysis of T cell subsets. PBMCs were stained with anti-CD4 PE-cy7, anti-CD25 APC, anti-IFN-γ FITC, anti-IL-17 PE, anti-IL-4 APC and anti-Foxp3 FITC. CD4+ cells were gated for further analysis. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of Golgi Stop. The percentage of Target cells was measured by flowcytometry. The frequency (%) of Lymphocyte/Leukocyte cells, CD4 + T/Lymphocyte cells, IL-17+/CD4 + T cells, IFN-γ + /CD4 + T cells, IL-4 + /CD4 + T cells (A) and CD25 + FOXP3 + /CD4 + T cells (B) in patients before KT, patients at 1month after KT and patients at 3 month after KT. After surface staining with anti-CD4, CD45 and CCR7 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with IL-17 mAbs. T naïve /CD4 + T (CD45RA + CCR7 + /CD4 + Tcells), IL-17 + /T naïve , T CM /CD4 + T (CD45RA − CCR7 + /CD4 + Tcells), IL-17 + /T CM + and T EM /CD4 + T (CD45RA − CCR7 − /CD4 + Tcells), IL-17 + /T EM + (C).
Figure Legend Snippet: Flow cytometric analysis of T cell subsets. PBMCs were stained with anti-CD4 PE-cy7, anti-CD25 APC, anti-IFN-γ FITC, anti-IL-17 PE, anti-IL-4 APC and anti-Foxp3 FITC. CD4+ cells were gated for further analysis. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of Golgi Stop. The percentage of Target cells was measured by flowcytometry. The frequency (%) of Lymphocyte/Leukocyte cells, CD4 + T/Lymphocyte cells, IL-17+/CD4 + T cells, IFN-γ + /CD4 + T cells, IL-4 + /CD4 + T cells (A) and CD25 + FOXP3 + /CD4 + T cells (B) in patients before KT, patients at 1month after KT and patients at 3 month after KT. After surface staining with anti-CD4, CD45 and CCR7 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with IL-17 mAbs. T naïve /CD4 + T (CD45RA + CCR7 + /CD4 + Tcells), IL-17 + /T naïve , T CM /CD4 + T (CD45RA − CCR7 + /CD4 + Tcells), IL-17 + /T CM + and T EM /CD4 + T (CD45RA − CCR7 − /CD4 + Tcells), IL-17 + /T EM + (C).

Techniques Used: Flow Cytometry, Staining, Ex Vivo

39) Product Images from "Cross-talk between iNKT cells and CD8 T cells in the spleen requires the IL-4/CCL17 axis for the generation of short-lived effector cells"

Article Title: Cross-talk between iNKT cells and CD8 T cells in the spleen requires the IL-4/CCL17 axis for the generation of short-lived effector cells

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1913491116

CCR4 and CXCR3 expressions increase over time at the cell surface upon vaccination and play a role in early T cell activation. CD8 + OT-I T cells were isolated, labeled with CFR dye, and adoptively transferred prior vaccination. The next day, nanovaccines containing OVA and α-Galcer were intravenously administered in mice. At different time points, mice were killed, spleens harvested, stained, and analyzed by flow cytometry. ( A ) Representative expression of CXCR3, CCR4, and CD69 on OT-I T cells in control or vaccinated mice at 6 and 24 h. Results are representative of 2 independent experiments. ( B and C ) One day prior to vaccination, anti-CXCR3 blocking antibodies or CCR4 antagonist was injected intraperitoneally. ( B ) Six hours after nanovaccine administration, spleens were harvested and CD69 expression on CFR-labeled OT-I T cells were evaluated by flow cytometry. Representative experiment ( Left ). Percentage of inhibition is indicated as the percentage of CD69 + OT-I T cells in blocking antibody-treated cells relative to the percentage of CD69 + OT-I T cells in vehicle-treated cells ( Right ). Pool from 3 independent experiments for CXCR3 blockade and 2 independent experiments for CCR4 blockade. Statistical analysis by t test: * P
Figure Legend Snippet: CCR4 and CXCR3 expressions increase over time at the cell surface upon vaccination and play a role in early T cell activation. CD8 + OT-I T cells were isolated, labeled with CFR dye, and adoptively transferred prior vaccination. The next day, nanovaccines containing OVA and α-Galcer were intravenously administered in mice. At different time points, mice were killed, spleens harvested, stained, and analyzed by flow cytometry. ( A ) Representative expression of CXCR3, CCR4, and CD69 on OT-I T cells in control or vaccinated mice at 6 and 24 h. Results are representative of 2 independent experiments. ( B and C ) One day prior to vaccination, anti-CXCR3 blocking antibodies or CCR4 antagonist was injected intraperitoneally. ( B ) Six hours after nanovaccine administration, spleens were harvested and CD69 expression on CFR-labeled OT-I T cells were evaluated by flow cytometry. Representative experiment ( Left ). Percentage of inhibition is indicated as the percentage of CD69 + OT-I T cells in blocking antibody-treated cells relative to the percentage of CD69 + OT-I T cells in vehicle-treated cells ( Right ). Pool from 3 independent experiments for CXCR3 blockade and 2 independent experiments for CCR4 blockade. Statistical analysis by t test: * P

Techniques Used: Activation Assay, Isolation, Labeling, Mouse Assay, Staining, Flow Cytometry, Expressing, Blocking Assay, Injection, Inhibition

NKT cells make long-lasting contacts together with OT-I T cells and DC at late stages of activation. ( A ) Single-cell suspension of splenocytes from CXCR6-GFP mice were stained with CD8, CD3, and NK1.1 antibodies, and Cd1d/α-Galcer dextramer and analyzed by flow cytometry. ( B and F ) CD8 + OT-I T cells were purified, labeled with CFR dye, and adoptively transferred in CXCR6-GFP mice prior to vaccination. One day later, nanovaccines containing OVA and α-Galcer were intravenously administered in CXCR6-GFP mice. At 0, 6, and 24 h, mice were killed, spleens harvested, and fixed in order to perform cryosections (20 μm) for subsequent immunofluorescent staining. Cryosections were stained with either anti-CD169 or anti-CD11c antibodies. ( B ) Representative images of the CD169 (red) and CXCR6-GFP (green) costaining at the different time points. (Scale bars, 50 µm.) ( C ) Percentage of the CXCR6 hi cell localized in the WP at the different time points. ( D ) Percentage of the clustering of CXCR6 hi cells at the different time points. ( E ) Percentage of OT-I T cells in close contacts (distance inferior to 3 µm) with CXCR6 hi cells in the MZ/RP or in the WP at the different time points. ( F ) Representative images of OT-I (white), CD11c (red), and CXCR6-GFP (green) costaining at 24 h in the WP. (Scale bar, 10 µm.) ( G ) CD8 + OT-I T cells were purified, labeled with CFR dye and adoptively transferred in CXCR6-GFP mice prior to vaccination. Sixteen hours later, nanovaccines containing OVA and α-Galcer or only α-Galcer were intravenously administered in CXCR6-GFP mice. Six and 24 h later, mice were killed, spleens harvested, and embedded in a low-melting agarose gel. Thick sections of 500 μm were performed using a vibratome. and stained with anti-CD169 and anti-CD11c antibodies. Live imaging was performed using a spinning-disk microscope equipped with a thermostated chamber and perfused at a rate of 0.8 mL/min with medium bubbled with 95% O 2 and 5% CO 2 . OT-I and NKT contacts were evaluated on movies lasting 30 min. ( H ) From analyzed images for the 24-h time point, the positions of NKT and OT-I T cells in the WP were extracted. For each picture, 20 runs of simulation were performed in order to randomly change the location of NKT cells. The closest distance between OT-I and NKT cells was measured for every OT-I T cell and the mean of the closest distance between these 2 cell types was calculated and then compared to the 1 from the original picture. ( I ) One day prior to vaccination, anti-CXCR3 blocking antibodies or CCR4 antagonist was injected intraperitoneally into CXCR6-GFP mice. Then, nanovaccines containing OVA and α-Galcer were intravenously administered, and spleens were harvested 24 h after vaccination, and fixed in order to perform cryosections (20 μm) for subsequent immunofluorescent staining with anti-CD169 antibody. NKT cell density (cells/mm 2 ) in the WP was calculated. Results involve at least 2 mice per condition and time point. Statistical analysis by 1-way ANOVA test for C , D , G , and I , and by t test for E and H : * P
Figure Legend Snippet: NKT cells make long-lasting contacts together with OT-I T cells and DC at late stages of activation. ( A ) Single-cell suspension of splenocytes from CXCR6-GFP mice were stained with CD8, CD3, and NK1.1 antibodies, and Cd1d/α-Galcer dextramer and analyzed by flow cytometry. ( B and F ) CD8 + OT-I T cells were purified, labeled with CFR dye, and adoptively transferred in CXCR6-GFP mice prior to vaccination. One day later, nanovaccines containing OVA and α-Galcer were intravenously administered in CXCR6-GFP mice. At 0, 6, and 24 h, mice were killed, spleens harvested, and fixed in order to perform cryosections (20 μm) for subsequent immunofluorescent staining. Cryosections were stained with either anti-CD169 or anti-CD11c antibodies. ( B ) Representative images of the CD169 (red) and CXCR6-GFP (green) costaining at the different time points. (Scale bars, 50 µm.) ( C ) Percentage of the CXCR6 hi cell localized in the WP at the different time points. ( D ) Percentage of the clustering of CXCR6 hi cells at the different time points. ( E ) Percentage of OT-I T cells in close contacts (distance inferior to 3 µm) with CXCR6 hi cells in the MZ/RP or in the WP at the different time points. ( F ) Representative images of OT-I (white), CD11c (red), and CXCR6-GFP (green) costaining at 24 h in the WP. (Scale bar, 10 µm.) ( G ) CD8 + OT-I T cells were purified, labeled with CFR dye and adoptively transferred in CXCR6-GFP mice prior to vaccination. Sixteen hours later, nanovaccines containing OVA and α-Galcer or only α-Galcer were intravenously administered in CXCR6-GFP mice. Six and 24 h later, mice were killed, spleens harvested, and embedded in a low-melting agarose gel. Thick sections of 500 μm were performed using a vibratome. and stained with anti-CD169 and anti-CD11c antibodies. Live imaging was performed using a spinning-disk microscope equipped with a thermostated chamber and perfused at a rate of 0.8 mL/min with medium bubbled with 95% O 2 and 5% CO 2 . OT-I and NKT contacts were evaluated on movies lasting 30 min. ( H ) From analyzed images for the 24-h time point, the positions of NKT and OT-I T cells in the WP were extracted. For each picture, 20 runs of simulation were performed in order to randomly change the location of NKT cells. The closest distance between OT-I and NKT cells was measured for every OT-I T cell and the mean of the closest distance between these 2 cell types was calculated and then compared to the 1 from the original picture. ( I ) One day prior to vaccination, anti-CXCR3 blocking antibodies or CCR4 antagonist was injected intraperitoneally into CXCR6-GFP mice. Then, nanovaccines containing OVA and α-Galcer were intravenously administered, and spleens were harvested 24 h after vaccination, and fixed in order to perform cryosections (20 μm) for subsequent immunofluorescent staining with anti-CD169 antibody. NKT cell density (cells/mm 2 ) in the WP was calculated. Results involve at least 2 mice per condition and time point. Statistical analysis by 1-way ANOVA test for C , D , G , and I , and by t test for E and H : * P

Techniques Used: Activation Assay, Mouse Assay, Staining, Flow Cytometry, Purification, Labeling, Agarose Gel Electrophoresis, Imaging, Microscopy, Blocking Assay, Injection

IL-4/CCL17 axis promotes the generation of short-lived effector cells. ( A ) nanovaccines containing OVA and α-Galcer or TLR-L were intravenously administered in mice. At different time points, mice were killed, sera were collected, and ELISA for IL-4 was performed. ( B ) CD8 + OT-I T cells were isolated, labeled with CFR, and adoptively transferred prior to vaccination. The next day, nanovaccines containing OVA and α-Galcer were intravenously administered in mice. At 6 or 24 h, mice were killed, spleens harvested, stained, and analyzed by flow cytometry. Representative expression of pSTAT6 on OT-I T cells in control or vaccinated mice at 6 and 24 h. Results are representative of 2 independent experiments. ( C and D ) CD8 + OT-I T cells were isolated, labeled with CFR, and adoptively transferred prior to vaccination. One day prior to vaccination, isotype or anti–IL-4 blocking antibodies were injected intraperitoneally. The next day, nanovaccines containing OVA and α-Galcer were intravenously administered in mice and spleens from the 24-h time point were harvested. ( C ) Expression of CCR4 on OT-I T cells was evaluated by flow cytometry. ( D ) Expression of CCL17 in spleen cryosections (20 µm) was evaluated by confocal microscopy. (Scale bars, 50 µm.) ( E – H ) One-thousand CD8 + OT-I T cells were adoptively transferred prior to vaccination into CD45.1 mice. One day prior to vaccination, isotype, anti-CXCR3 ( E and F ), or anti-CCL17 or IL-4 antibodies ( G and H ) were injected intraperitoneally. The next day, nanovaccines containing OVA and α-Galcer were intravenously administered in mice and 10 d later spleens were harvested and analyzed by flow cytometry. MPEC were defined as CD127 hi KLGR1 − cells and SLEC as CD127 lo KLGR1 + cells. Pool from 2 independent experiments with at least 5 mice per condition. Statistical analysis by 1-way ANOVA test for A and t test for C – H : * P
Figure Legend Snippet: IL-4/CCL17 axis promotes the generation of short-lived effector cells. ( A ) nanovaccines containing OVA and α-Galcer or TLR-L were intravenously administered in mice. At different time points, mice were killed, sera were collected, and ELISA for IL-4 was performed. ( B ) CD8 + OT-I T cells were isolated, labeled with CFR, and adoptively transferred prior to vaccination. The next day, nanovaccines containing OVA and α-Galcer were intravenously administered in mice. At 6 or 24 h, mice were killed, spleens harvested, stained, and analyzed by flow cytometry. Representative expression of pSTAT6 on OT-I T cells in control or vaccinated mice at 6 and 24 h. Results are representative of 2 independent experiments. ( C and D ) CD8 + OT-I T cells were isolated, labeled with CFR, and adoptively transferred prior to vaccination. One day prior to vaccination, isotype or anti–IL-4 blocking antibodies were injected intraperitoneally. The next day, nanovaccines containing OVA and α-Galcer were intravenously administered in mice and spleens from the 24-h time point were harvested. ( C ) Expression of CCR4 on OT-I T cells was evaluated by flow cytometry. ( D ) Expression of CCL17 in spleen cryosections (20 µm) was evaluated by confocal microscopy. (Scale bars, 50 µm.) ( E – H ) One-thousand CD8 + OT-I T cells were adoptively transferred prior to vaccination into CD45.1 mice. One day prior to vaccination, isotype, anti-CXCR3 ( E and F ), or anti-CCL17 or IL-4 antibodies ( G and H ) were injected intraperitoneally. The next day, nanovaccines containing OVA and α-Galcer were intravenously administered in mice and 10 d later spleens were harvested and analyzed by flow cytometry. MPEC were defined as CD127 hi KLGR1 − cells and SLEC as CD127 lo KLGR1 + cells. Pool from 2 independent experiments with at least 5 mice per condition. Statistical analysis by 1-way ANOVA test for A and t test for C – H : * P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Isolation, Labeling, Staining, Flow Cytometry, Expressing, Blocking Assay, Injection, Confocal Microscopy

40) Product Images from "Serine/threonine phosphatase PP2A is essential for optimal B cell function"

Article Title: Serine/threonine phosphatase PP2A is essential for optimal B cell function

Journal: JCI Insight

doi: 10.1172/jci.insight.130655

PP2A is critical for B cell activation, differentiation, and Ig production in vivo. ( A ) Dot plots show the percentages of spontaneous germinal center B cells (GC-CD19 + FAS + PNA + ) and T follicular helper cells (Tfh-CD4 + CXCR5 hi PD1 hi ) in spleens of the indicated 24-week-old mice ( n = 6 mice per group for 2 independent experiments). ( B and C ) Mice were immunized i.p. with 0.2 mL/mouse SRBC. ( B ) IHC staining of frozen spleen sections of the indicated mice ( n = 3 mice per group in 2 independent experiments). Bar graphs show the quantification (percentage) of follicles with germinal centers in total splenic follicles from the indicated mice ( n = 3 mice per group in 2 independent experiments). ( C ) Flow cytometry analysis (%) of germinal center B cells (GC-CD19 + FAS + PNA + ), T follicular helper cells (Tfh-CD4 + CXCR5 hi PD1 hi ), and IgG plasma cells (PC-CD19 + IgG + CD138 + ) in the spleens of the indicated mice after SRBC immunization. ( D and E ) Indicated mice were i.p. immunized with 100 μg/mouse NP-Ficoll for 5 days or 100 μg/mouse NP-CGG in 5% alum for 14 days. ( D ) ELISA analysis for high affinity (NP-7) or low affinity (NP-41) for NP antigen-specific IgM in the serum of 12-week-old flox/flox mice compared with control mice 5 days after immunization with T-independent antigen NP-Ficoll ( n = 3 per group in 2 independent experiments). ( E ) ELISA analysis of high affinity (NP-7) or all affinity (NP-41) NP antigen-specific IgG in the serum of 12-week-old flox/flox mice compared with control mice 14 days after immunization with T-dependent antigen NP-CGG ( n = 3 per group in 2 independent experiments). Paired t test, ** P
Figure Legend Snippet: PP2A is critical for B cell activation, differentiation, and Ig production in vivo. ( A ) Dot plots show the percentages of spontaneous germinal center B cells (GC-CD19 + FAS + PNA + ) and T follicular helper cells (Tfh-CD4 + CXCR5 hi PD1 hi ) in spleens of the indicated 24-week-old mice ( n = 6 mice per group for 2 independent experiments). ( B and C ) Mice were immunized i.p. with 0.2 mL/mouse SRBC. ( B ) IHC staining of frozen spleen sections of the indicated mice ( n = 3 mice per group in 2 independent experiments). Bar graphs show the quantification (percentage) of follicles with germinal centers in total splenic follicles from the indicated mice ( n = 3 mice per group in 2 independent experiments). ( C ) Flow cytometry analysis (%) of germinal center B cells (GC-CD19 + FAS + PNA + ), T follicular helper cells (Tfh-CD4 + CXCR5 hi PD1 hi ), and IgG plasma cells (PC-CD19 + IgG + CD138 + ) in the spleens of the indicated mice after SRBC immunization. ( D and E ) Indicated mice were i.p. immunized with 100 μg/mouse NP-Ficoll for 5 days or 100 μg/mouse NP-CGG in 5% alum for 14 days. ( D ) ELISA analysis for high affinity (NP-7) or low affinity (NP-41) for NP antigen-specific IgM in the serum of 12-week-old flox/flox mice compared with control mice 5 days after immunization with T-independent antigen NP-Ficoll ( n = 3 per group in 2 independent experiments). ( E ) ELISA analysis of high affinity (NP-7) or all affinity (NP-41) NP antigen-specific IgG in the serum of 12-week-old flox/flox mice compared with control mice 14 days after immunization with T-dependent antigen NP-CGG ( n = 3 per group in 2 independent experiments). Paired t test, ** P

Techniques Used: Activation Assay, In Vivo, Mouse Assay, Immunohistochemistry, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Related Articles

Flow Cytometry:

Article Title: Blimp1 Prevents Methylation of Foxp3 and Loss of Regulatory T Cell Identity at Sites of Inflammation
Article Snippet: .. Antibodies and flow cytometry Cell suspensions from lymphoid organs were stained with fluorochrome-conjugated anti-mouse CD4 (RM4-5), CD25 (PC61.5), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD62L (MEL-14), CD90.1 (OX-7), GITR (CD357) (DTA-1), Ki67 (16A8), CD126 (D7715A7) and KLRG1 (2F1), which were purchased from either Biolegend, eBioscience or BD Biosciences. .. For dead cell exclusion, LIVE/DEAD® fixable Near-IR stain kit (Invitrogen) was used.

Article Title: Systemic ST6Gal-1 Is a Pro-survival Factor for Murine Transitional B Cells
Article Snippet: .. Antibodies For flow cytometry, anti-B220-PE/Cy7 (RA3-6B2), anti-CD19-BV510 (GD5), anti-IgD-PE (11-26c.2a), anti-CD23-APC/Cy7 (B3B4), anti-IgM-APC (RMM-1), anti-CD21-PerCP/Cy5.5 (7E9), and anti-CD24-PE (30-F1) were purchased from Biolegend. .. For magnetic cell separation, biotinylated anti-IgM (RMM-1), anti-B220 (RA3-6B2), anti-Gr1 (RB6-8C5), anti-CD23 (B3B4), and anti-CD3e antibody (145-2C11) were purchased from BD Pharmingen.

Cytometry:

Article Title: Blimp1 Prevents Methylation of Foxp3 and Loss of Regulatory T Cell Identity at Sites of Inflammation
Article Snippet: .. Antibodies and flow cytometry Cell suspensions from lymphoid organs were stained with fluorochrome-conjugated anti-mouse CD4 (RM4-5), CD25 (PC61.5), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD62L (MEL-14), CD90.1 (OX-7), GITR (CD357) (DTA-1), Ki67 (16A8), CD126 (D7715A7) and KLRG1 (2F1), which were purchased from either Biolegend, eBioscience or BD Biosciences. .. For dead cell exclusion, LIVE/DEAD® fixable Near-IR stain kit (Invitrogen) was used.

Article Title: Systemic ST6Gal-1 Is a Pro-survival Factor for Murine Transitional B Cells
Article Snippet: .. Antibodies For flow cytometry, anti-B220-PE/Cy7 (RA3-6B2), anti-CD19-BV510 (GD5), anti-IgD-PE (11-26c.2a), anti-CD23-APC/Cy7 (B3B4), anti-IgM-APC (RMM-1), anti-CD21-PerCP/Cy5.5 (7E9), and anti-CD24-PE (30-F1) were purchased from Biolegend. .. For magnetic cell separation, biotinylated anti-IgM (RMM-1), anti-B220 (RA3-6B2), anti-Gr1 (RB6-8C5), anti-CD23 (B3B4), and anti-CD3e antibody (145-2C11) were purchased from BD Pharmingen.

Construct:

Article Title: Immune checkpoints PVR and PVRL2 are prognostic markers in AML and their blockade represents a new therapeutic option
Article Snippet: .. Two hundred microliter of cell suspension was plated in triplicates in 96-well plates in corresponding cell culture medium and incubated with or without 4 µg/mL blocking anti-PVR (clone D171, NeoMarkers), 25 µL/mL blocking anti-PVRL2 (clone L14 [ ]), or 50 µg/mL blocking anti-TIGIT (Clone #A15153G, Biolegend) antibodies in the presence or absence of the BiTE® antibody construct AMG 330 (AMGEN Inc.) at 0.1 ng/mL. .. Assessment of specific lysis of the target cells was performed after 24 h of incubation by measuring the 7AAD (BD Biosciences) staining of gated CMFDA positive cells in flow cytometry.

Purification:

Article Title: Murine DX5+NKT Cells Display Their Cytotoxic and Proapoptotic Potentials against Colitis-Inducing CD4+CD62Lhigh T Cells through Fas Ligand
Article Snippet: .. For FasL blocking (Kayagaki, Yamaguchi et al. 1997), DX5+ NKT cells were preincubated with either 50 μ g/ml purified mouse-anti-FasL (clone: MFL4; BioLegend, Cambridge, UK) or 50 μ g/ml isotype control for 1 h and then cocultures were set up with CD4+ CD62Lhigh cells as indicated above. ..

Incubation:

Article Title: CCR4-dependent reduction in the number and suppressor function of CD4+Foxp3+ cells augments IFN-γ-mediated pulmonary inflammation and aggravates tuberculosis pathogenesis
Article Snippet: .. After washing, the cells were incubated for 20 min at 4 °C with supernatant from 2.4G2 cells, followed by incubation with anti-CD4, anti-IFN-γ (XMG1.2), anti-IL-17 (TC11-18H10) (BioLegend, San Diego, CA, USA), anti-IL-4 (11B11) and anti-IL-10 (JES5− 16E3) (BD Biosciences, San Jose, CA, USA) antibodies for 30 min at 4 °C in total darkness. .. Data on cells were acquired by flow cytometry using a BD FACSCanto II instrument (BD Bioscience, Franklin Lakes, NJ, USA).

Article Title: Immune checkpoints PVR and PVRL2 are prognostic markers in AML and their blockade represents a new therapeutic option
Article Snippet: .. Two hundred microliter of cell suspension was plated in triplicates in 96-well plates in corresponding cell culture medium and incubated with or without 4 µg/mL blocking anti-PVR (clone D171, NeoMarkers), 25 µL/mL blocking anti-PVRL2 (clone L14 [ ]), or 50 µg/mL blocking anti-TIGIT (Clone #A15153G, Biolegend) antibodies in the presence or absence of the BiTE® antibody construct AMG 330 (AMGEN Inc.) at 0.1 ng/mL. .. Assessment of specific lysis of the target cells was performed after 24 h of incubation by measuring the 7AAD (BD Biosciences) staining of gated CMFDA positive cells in flow cytometry.

other:

Article Title: Actin stabilizer TAGLN2 potentiates adoptive T cell therapy by boosting the inside-out costimulation via lymphocyte function-associated antigen-1
Article Snippet: Anti-mouse LFA-1, fluorescein isothiocyanate (FITC)-conjugated anti-mouse ICAM-1, and FITC-rat IgG1 isotype control antibodies were purchased from Biolegend (San Diego, CA).

Cell Culture:

Article Title: Immune checkpoints PVR and PVRL2 are prognostic markers in AML and their blockade represents a new therapeutic option
Article Snippet: .. Two hundred microliter of cell suspension was plated in triplicates in 96-well plates in corresponding cell culture medium and incubated with or without 4 µg/mL blocking anti-PVR (clone D171, NeoMarkers), 25 µL/mL blocking anti-PVRL2 (clone L14 [ ]), or 50 µg/mL blocking anti-TIGIT (Clone #A15153G, Biolegend) antibodies in the presence or absence of the BiTE® antibody construct AMG 330 (AMGEN Inc.) at 0.1 ng/mL. .. Assessment of specific lysis of the target cells was performed after 24 h of incubation by measuring the 7AAD (BD Biosciences) staining of gated CMFDA positive cells in flow cytometry.

Staining:

Article Title: Blimp1 Prevents Methylation of Foxp3 and Loss of Regulatory T Cell Identity at Sites of Inflammation
Article Snippet: .. Antibodies and flow cytometry Cell suspensions from lymphoid organs were stained with fluorochrome-conjugated anti-mouse CD4 (RM4-5), CD25 (PC61.5), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD62L (MEL-14), CD90.1 (OX-7), GITR (CD357) (DTA-1), Ki67 (16A8), CD126 (D7715A7) and KLRG1 (2F1), which were purchased from either Biolegend, eBioscience or BD Biosciences. .. For dead cell exclusion, LIVE/DEAD® fixable Near-IR stain kit (Invitrogen) was used.

Article Title: Endothelial progenitor cell-conditioned medium promotes angiogenesis and is neuroprotective after spinal cord injury
Article Snippet: .. Single-cell suspensions were prepared in phosphate-buffered saline (PBS) and blocked with anti-mouse CD16/32 for 10 minutes, and thereafter stained with rat anti-mouse F4/80, CD11b, CD86 or CD206 antibody (1 µg per 1 × 106 cells; BioLegend, CA, USA) for 20 minutes on ice. .. After three PBS washes, samples were analyzed with the BD FACSverse flow cytometry system (BD, Franklin Lakes, NJ, USA).

Blocking Assay:

Article Title: Immune checkpoints PVR and PVRL2 are prognostic markers in AML and their blockade represents a new therapeutic option
Article Snippet: .. Two hundred microliter of cell suspension was plated in triplicates in 96-well plates in corresponding cell culture medium and incubated with or without 4 µg/mL blocking anti-PVR (clone D171, NeoMarkers), 25 µL/mL blocking anti-PVRL2 (clone L14 [ ]), or 50 µg/mL blocking anti-TIGIT (Clone #A15153G, Biolegend) antibodies in the presence or absence of the BiTE® antibody construct AMG 330 (AMGEN Inc.) at 0.1 ng/mL. .. Assessment of specific lysis of the target cells was performed after 24 h of incubation by measuring the 7AAD (BD Biosciences) staining of gated CMFDA positive cells in flow cytometry.

Article Title: Murine DX5+NKT Cells Display Their Cytotoxic and Proapoptotic Potentials against Colitis-Inducing CD4+CD62Lhigh T Cells through Fas Ligand
Article Snippet: .. For FasL blocking (Kayagaki, Yamaguchi et al. 1997), DX5+ NKT cells were preincubated with either 50 μ g/ml purified mouse-anti-FasL (clone: MFL4; BioLegend, Cambridge, UK) or 50 μ g/ml isotype control for 1 h and then cocultures were set up with CD4+ CD62Lhigh cells as indicated above. ..

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91
    BioLegend anti igd pe
    Expression of ST6Gal-1, α2,6-sialyl ligands, and CD22 in B cells. (A) Bone marrow immature (IM), <t>IgM-high,</t> and mature (BMM), as well as splenic <t>IgD-/CD21-,</t> IgD+/CD21+, marginal zone (MZ), and follicular (FO) populations were isolated by fluorescence activated cell sorting (FACS) ( > 94% purity). RT-qPCR was performed for ST6Gal-1 transcripts, and representative results of three independent experiments shown relative to β2-microglobulin ( n = 3). Western blot analysis of protein levels in splenic populations is quantified relative to β-actin ( n = 3). (B) Mean SNA reactivity is shown for bone marrow and splenic B cell subsets ( n = 5 or 10). (C) Frequency of cell surface CD22 expression in BM and splenic B cell populations ( n = 5). (D) Relative RNA expression of ST6Gal-1 and SNA reactivity are compared, with standard deviations shown in both dimensions, and arrows indicating select developmental steps. (E) CD22 expression and SNA reactivity is compared, with standard deviations of measurement shown in both dimensions. Arrows indicate sequence of B cell development.
    Anti Igd Pe, supplied by BioLegend, used in various techniques. Bioz Stars score: 91/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti igd pe/product/BioLegend
    Average 91 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    anti igd pe - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    92
    BioLegend phycoerythrin pe conjugated anti cd45
    TRPA1 inhibition reduces M2 macrophage polarization. (A) Immunofluorescence analysis of M2 macrophages (anti-CD206) in mice at 4 weeks after TAC surgery (n = 7). (B) Flow cytometry analysis of M2 macrophage <t>(CD45</t> + F4/80 + CD206 + ) expression in mice at 4 weeks after TAC surgery (n = 4). (C) RT-PCR analysis of interleukin-4 (IL-4), IL-10 and transforming growth factor-β (TGF-β) expression in bone marrow–derived macrophages (BMDMs) cultured with and without Ang II (n = 8). *P
    Phycoerythrin Pe Conjugated Anti Cd45, supplied by BioLegend, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phycoerythrin pe conjugated anti cd45/product/BioLegend
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phycoerythrin pe conjugated anti cd45 - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    93
    BioLegend rat anti mouse f4 80
    Effects of EPC-CM on inflammatory cytokine levels in vitro and in vivo . (A) Representative flow cytometry data showing the effect of EPC-CM on BMDMs. (a) Mature BMDMs were defined as CD11b + <t>/F4/80</t> + subpopulations (upper right), with the purity displayed as percentage of the parent population. (b) Control BMDMs were incubated with CD11b and F4/80 antibody and used to set up the gate. (c) Control BMDMs without LPS stimulation were incubated with anti-rat CD11b, F4/80, CD86 and CD206 antibodies. M1 macrophages are CD11b + /F4/80 + /CD86 + /CD206 − (Q1), whereas M2 macrophages are CD11b + /F4/80 + /CD86 − /CD206 + (Q3). (d) BMDMs treated with LPS. (e) BMDMs cultured with Con-M and simultaneously stimulated with LPS. (f) BMDMs cultured with EPC-CM and stimulated with LPS. (B) Quantitation of M1 and M2 cells among the different groups. Compared with the Con-M group, EPC-CM significantly reduced M1 activation, while M2 cells remained relatively unchanged. (C) mRNA expression levels of inflammatory cytokines (optical density ratio) among groups. (D) Immunofluorescence staining for CD86 (M1 marker) and CD206 (M2 marker) in the epicenter 7 days after SCI ( n = 5 per group; green: CD86; red: CD206; blue: DAPI). (E) Quantification of CD86- and CD206-positive cells at 7 days after SCI. ** P
    Rat Anti Mouse F4 80, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rat anti mouse f4 80/product/BioLegend
    Average 93 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    rat anti mouse f4 80 - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    93
    BioLegend fluorochrome conjugated anti mouse cd4
    Blimp1 Governs Treg Cell Identity in CNS Treg Cells Mixed bone marrow chimeras (MBMCs) were generated by reconstituting Rag1 −/− hosts with (CD45.1, wild-type) and Blimp1 ΔFoxp3 (CD45.2) bone marrow at a ratio of 1:1. The mixed bone marrow chimeras were immunized with MOG(35-55) in CFA to induce EAE. The mice were analyzed at the peak of EAE. (A) Chimerism between wild-type and Blimp1 ΔFoxp3 cells in the live <t>CD4</t> + gate of spleen and CNS mononuclear cells and analysis of Foxp3 expression by flow cytometry in control (wild-type) Treg cells and Blimp1 ΔFoxp3 Treg cells isolated from the mixed bone marrow chimeras at the peak of EAE. Data are representative and summarized from three biological replicates. Symbols depict individual mice (bars, mean ± SD). Student’s t test ( ∗ p
    Fluorochrome Conjugated Anti Mouse Cd4, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fluorochrome conjugated anti mouse cd4/product/BioLegend
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    fluorochrome conjugated anti mouse cd4 - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    Image Search Results


    Expression of ST6Gal-1, α2,6-sialyl ligands, and CD22 in B cells. (A) Bone marrow immature (IM), IgM-high, and mature (BMM), as well as splenic IgD-/CD21-, IgD+/CD21+, marginal zone (MZ), and follicular (FO) populations were isolated by fluorescence activated cell sorting (FACS) ( > 94% purity). RT-qPCR was performed for ST6Gal-1 transcripts, and representative results of three independent experiments shown relative to β2-microglobulin ( n = 3). Western blot analysis of protein levels in splenic populations is quantified relative to β-actin ( n = 3). (B) Mean SNA reactivity is shown for bone marrow and splenic B cell subsets ( n = 5 or 10). (C) Frequency of cell surface CD22 expression in BM and splenic B cell populations ( n = 5). (D) Relative RNA expression of ST6Gal-1 and SNA reactivity are compared, with standard deviations shown in both dimensions, and arrows indicating select developmental steps. (E) CD22 expression and SNA reactivity is compared, with standard deviations of measurement shown in both dimensions. Arrows indicate sequence of B cell development.

    Journal: Frontiers in Immunology

    Article Title: Systemic ST6Gal-1 Is a Pro-survival Factor for Murine Transitional B Cells

    doi: 10.3389/fimmu.2018.02150

    Figure Lengend Snippet: Expression of ST6Gal-1, α2,6-sialyl ligands, and CD22 in B cells. (A) Bone marrow immature (IM), IgM-high, and mature (BMM), as well as splenic IgD-/CD21-, IgD+/CD21+, marginal zone (MZ), and follicular (FO) populations were isolated by fluorescence activated cell sorting (FACS) ( > 94% purity). RT-qPCR was performed for ST6Gal-1 transcripts, and representative results of three independent experiments shown relative to β2-microglobulin ( n = 3). Western blot analysis of protein levels in splenic populations is quantified relative to β-actin ( n = 3). (B) Mean SNA reactivity is shown for bone marrow and splenic B cell subsets ( n = 5 or 10). (C) Frequency of cell surface CD22 expression in BM and splenic B cell populations ( n = 5). (D) Relative RNA expression of ST6Gal-1 and SNA reactivity are compared, with standard deviations shown in both dimensions, and arrows indicating select developmental steps. (E) CD22 expression and SNA reactivity is compared, with standard deviations of measurement shown in both dimensions. Arrows indicate sequence of B cell development.

    Article Snippet: Antibodies For flow cytometry, anti-B220-PE/Cy7 (RA3-6B2), anti-CD19-BV510 (GD5), anti-IgD-PE (11-26c.2a), anti-CD23-APC/Cy7 (B3B4), anti-IgM-APC (RMM-1), anti-CD21-PerCP/Cy5.5 (7E9), and anti-CD24-PE (30-F1) were purchased from Biolegend.

    Techniques: Expressing, Isolation, Fluorescence, FACS, Quantitative RT-PCR, Western Blot, RNA Expression, Sequencing

    Cell non-autonomous ST6Gal-1 influences sialylation and abundance of early transitional B cell populations. CD45.1+ whole bone marrow cells from wild-type or St6gal1-KO mice were adoptively transferred to irradiated CD45.2+ hosts. Mice were allowed to recover for 6 weeks before analysis of bone marrow and splenic B cells. (A) SNA reactivity of bone marrow and splenic B cell subsets of CD45.1+ donor cells. (B) Frequencies of CD45.1+ IM, IgM-high, BMM, IgD-/CD21-, IgD+/CD21+, MZ, and FO B cells as a fraction of total CD45.1+ B cells ( n = 5). (C) Immunofluorescence microscopy staining anti-IgM (red) and anti-IgD (green) in chimeras. Splenic B cell populations indicated are identified accordingly - T1: IgM+/IgD-, extrafollicular; T2 and FO: IgM-variable/IgD+, follicular, MZ: IgM+/IgD-, marginal sinus. * P

    Journal: Frontiers in Immunology

    Article Title: Systemic ST6Gal-1 Is a Pro-survival Factor for Murine Transitional B Cells

    doi: 10.3389/fimmu.2018.02150

    Figure Lengend Snippet: Cell non-autonomous ST6Gal-1 influences sialylation and abundance of early transitional B cell populations. CD45.1+ whole bone marrow cells from wild-type or St6gal1-KO mice were adoptively transferred to irradiated CD45.2+ hosts. Mice were allowed to recover for 6 weeks before analysis of bone marrow and splenic B cells. (A) SNA reactivity of bone marrow and splenic B cell subsets of CD45.1+ donor cells. (B) Frequencies of CD45.1+ IM, IgM-high, BMM, IgD-/CD21-, IgD+/CD21+, MZ, and FO B cells as a fraction of total CD45.1+ B cells ( n = 5). (C) Immunofluorescence microscopy staining anti-IgM (red) and anti-IgD (green) in chimeras. Splenic B cell populations indicated are identified accordingly - T1: IgM+/IgD-, extrafollicular; T2 and FO: IgM-variable/IgD+, follicular, MZ: IgM+/IgD-, marginal sinus. * P

    Article Snippet: Antibodies For flow cytometry, anti-B220-PE/Cy7 (RA3-6B2), anti-CD19-BV510 (GD5), anti-IgD-PE (11-26c.2a), anti-CD23-APC/Cy7 (B3B4), anti-IgM-APC (RMM-1), anti-CD21-PerCP/Cy5.5 (7E9), and anti-CD24-PE (30-F1) were purchased from Biolegend.

    Techniques: Mouse Assay, Irradiation, Immunofluorescence, Microscopy, Staining

    TRPA1 inhibition reduces M2 macrophage polarization. (A) Immunofluorescence analysis of M2 macrophages (anti-CD206) in mice at 4 weeks after TAC surgery (n = 7). (B) Flow cytometry analysis of M2 macrophage (CD45 + F4/80 + CD206 + ) expression in mice at 4 weeks after TAC surgery (n = 4). (C) RT-PCR analysis of interleukin-4 (IL-4), IL-10 and transforming growth factor-β (TGF-β) expression in bone marrow–derived macrophages (BMDMs) cultured with and without Ang II (n = 8). *P

    Journal: EBioMedicine

    Article Title: TRPA1 inhibition ameliorates pressure overload-induced cardiac hypertrophy and fibrosis in mice

    doi: 10.1016/j.ebiom.2018.08.022

    Figure Lengend Snippet: TRPA1 inhibition reduces M2 macrophage polarization. (A) Immunofluorescence analysis of M2 macrophages (anti-CD206) in mice at 4 weeks after TAC surgery (n = 7). (B) Flow cytometry analysis of M2 macrophage (CD45 + F4/80 + CD206 + ) expression in mice at 4 weeks after TAC surgery (n = 4). (C) RT-PCR analysis of interleukin-4 (IL-4), IL-10 and transforming growth factor-β (TGF-β) expression in bone marrow–derived macrophages (BMDMs) cultured with and without Ang II (n = 8). *P

    Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated anti-CD206, allophycocyanin (APC)-conjugated anti-F4/80, phycoerythrin (PE)-conjugated anti-CD45 and FITC-conjugated anti-CD3 were purchased from BioLegend (San Diego, CA, USA).

    Techniques: Inhibition, Immunofluorescence, Mouse Assay, Flow Cytometry, Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Cell Culture

    TRPA1 inhibition prevents pressure overload-induced macrophage infiltration in cardiac tissues. (A) Immunohistochemical analysis of CD3 and CD68 in heart sections (n = 7, scale bar, 50 μm). (B–C) Flow cytometry analysis of CD45 + cells and T cells (CD45 + CD3 + ) in mice at 4 weeks after TAC surgery (n = 4). *P

    Journal: EBioMedicine

    Article Title: TRPA1 inhibition ameliorates pressure overload-induced cardiac hypertrophy and fibrosis in mice

    doi: 10.1016/j.ebiom.2018.08.022

    Figure Lengend Snippet: TRPA1 inhibition prevents pressure overload-induced macrophage infiltration in cardiac tissues. (A) Immunohistochemical analysis of CD3 and CD68 in heart sections (n = 7, scale bar, 50 μm). (B–C) Flow cytometry analysis of CD45 + cells and T cells (CD45 + CD3 + ) in mice at 4 weeks after TAC surgery (n = 4). *P

    Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated anti-CD206, allophycocyanin (APC)-conjugated anti-F4/80, phycoerythrin (PE)-conjugated anti-CD45 and FITC-conjugated anti-CD3 were purchased from BioLegend (San Diego, CA, USA).

    Techniques: Inhibition, Immunohistochemistry, Flow Cytometry, Cytometry, Mouse Assay

    Effects of EPC-CM on inflammatory cytokine levels in vitro and in vivo . (A) Representative flow cytometry data showing the effect of EPC-CM on BMDMs. (a) Mature BMDMs were defined as CD11b + /F4/80 + subpopulations (upper right), with the purity displayed as percentage of the parent population. (b) Control BMDMs were incubated with CD11b and F4/80 antibody and used to set up the gate. (c) Control BMDMs without LPS stimulation were incubated with anti-rat CD11b, F4/80, CD86 and CD206 antibodies. M1 macrophages are CD11b + /F4/80 + /CD86 + /CD206 − (Q1), whereas M2 macrophages are CD11b + /F4/80 + /CD86 − /CD206 + (Q3). (d) BMDMs treated with LPS. (e) BMDMs cultured with Con-M and simultaneously stimulated with LPS. (f) BMDMs cultured with EPC-CM and stimulated with LPS. (B) Quantitation of M1 and M2 cells among the different groups. Compared with the Con-M group, EPC-CM significantly reduced M1 activation, while M2 cells remained relatively unchanged. (C) mRNA expression levels of inflammatory cytokines (optical density ratio) among groups. (D) Immunofluorescence staining for CD86 (M1 marker) and CD206 (M2 marker) in the epicenter 7 days after SCI ( n = 5 per group; green: CD86; red: CD206; blue: DAPI). (E) Quantification of CD86- and CD206-positive cells at 7 days after SCI. ** P

    Journal: Neural Regeneration Research

    Article Title: Endothelial progenitor cell-conditioned medium promotes angiogenesis and is neuroprotective after spinal cord injury

    doi: 10.4103/1673-5374.232484

    Figure Lengend Snippet: Effects of EPC-CM on inflammatory cytokine levels in vitro and in vivo . (A) Representative flow cytometry data showing the effect of EPC-CM on BMDMs. (a) Mature BMDMs were defined as CD11b + /F4/80 + subpopulations (upper right), with the purity displayed as percentage of the parent population. (b) Control BMDMs were incubated with CD11b and F4/80 antibody and used to set up the gate. (c) Control BMDMs without LPS stimulation were incubated with anti-rat CD11b, F4/80, CD86 and CD206 antibodies. M1 macrophages are CD11b + /F4/80 + /CD86 + /CD206 − (Q1), whereas M2 macrophages are CD11b + /F4/80 + /CD86 − /CD206 + (Q3). (d) BMDMs treated with LPS. (e) BMDMs cultured with Con-M and simultaneously stimulated with LPS. (f) BMDMs cultured with EPC-CM and stimulated with LPS. (B) Quantitation of M1 and M2 cells among the different groups. Compared with the Con-M group, EPC-CM significantly reduced M1 activation, while M2 cells remained relatively unchanged. (C) mRNA expression levels of inflammatory cytokines (optical density ratio) among groups. (D) Immunofluorescence staining for CD86 (M1 marker) and CD206 (M2 marker) in the epicenter 7 days after SCI ( n = 5 per group; green: CD86; red: CD206; blue: DAPI). (E) Quantification of CD86- and CD206-positive cells at 7 days after SCI. ** P

    Article Snippet: Single-cell suspensions were prepared in phosphate-buffered saline (PBS) and blocked with anti-mouse CD16/32 for 10 minutes, and thereafter stained with rat anti-mouse F4/80, CD11b, CD86 or CD206 antibody (1 µg per 1 × 106 cells; BioLegend, CA, USA) for 20 minutes on ice.

    Techniques: In Vitro, In Vivo, Flow Cytometry, Cytometry, Incubation, Cell Culture, Quantitation Assay, Activation Assay, Expressing, Immunofluorescence, Staining, Marker

    Blimp1 Governs Treg Cell Identity in CNS Treg Cells Mixed bone marrow chimeras (MBMCs) were generated by reconstituting Rag1 −/− hosts with (CD45.1, wild-type) and Blimp1 ΔFoxp3 (CD45.2) bone marrow at a ratio of 1:1. The mixed bone marrow chimeras were immunized with MOG(35-55) in CFA to induce EAE. The mice were analyzed at the peak of EAE. (A) Chimerism between wild-type and Blimp1 ΔFoxp3 cells in the live CD4 + gate of spleen and CNS mononuclear cells and analysis of Foxp3 expression by flow cytometry in control (wild-type) Treg cells and Blimp1 ΔFoxp3 Treg cells isolated from the mixed bone marrow chimeras at the peak of EAE. Data are representative and summarized from three biological replicates. Symbols depict individual mice (bars, mean ± SD). Student’s t test ( ∗ p

    Journal: Cell Reports

    Article Title: Blimp1 Prevents Methylation of Foxp3 and Loss of Regulatory T Cell Identity at Sites of Inflammation

    doi: 10.1016/j.celrep.2019.01.070

    Figure Lengend Snippet: Blimp1 Governs Treg Cell Identity in CNS Treg Cells Mixed bone marrow chimeras (MBMCs) were generated by reconstituting Rag1 −/− hosts with (CD45.1, wild-type) and Blimp1 ΔFoxp3 (CD45.2) bone marrow at a ratio of 1:1. The mixed bone marrow chimeras were immunized with MOG(35-55) in CFA to induce EAE. The mice were analyzed at the peak of EAE. (A) Chimerism between wild-type and Blimp1 ΔFoxp3 cells in the live CD4 + gate of spleen and CNS mononuclear cells and analysis of Foxp3 expression by flow cytometry in control (wild-type) Treg cells and Blimp1 ΔFoxp3 Treg cells isolated from the mixed bone marrow chimeras at the peak of EAE. Data are representative and summarized from three biological replicates. Symbols depict individual mice (bars, mean ± SD). Student’s t test ( ∗ p

    Article Snippet: Antibodies and flow cytometry Cell suspensions from lymphoid organs were stained with fluorochrome-conjugated anti-mouse CD4 (RM4-5), CD25 (PC61.5), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD62L (MEL-14), CD90.1 (OX-7), GITR (CD357) (DTA-1), Ki67 (16A8), CD126 (D7715A7) and KLRG1 (2F1), which were purchased from either Biolegend, eBioscience or BD Biosciences.

    Techniques: Generated, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Isolation

    CNS Treg Cells are Stable and Express Blimp1 in Response to Proinflammatory Cytokines (A) Mononuclear cells were isolated from the CNS of EAE mice at the peak of disease and were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin to analyze the expression of IL-10, IL-17, and IFN-γ in Treg cells by flow cytometry. Mean of eight biological replicates ± SD, derived from three independent experiments. Symbols depict individual mice (bars, mean ± SD). (B) CD4 + Foxp3 + Treg cells were sorted from the CNS and spleen (SPL) of Foxp3 (GFP) reporter mice at the peak of EAE and subjected to RNA-seq. Principal-component analysis. (C) Foxp3 expression by intracellular staining of splenic Treg cells and CNS Treg cells at the peak of EAE. Mean of six biological replicates ± SD, derived from two independent experiments. Symbols depict individual mice (bars, mean ± SD), t test, p

    Journal: Cell Reports

    Article Title: Blimp1 Prevents Methylation of Foxp3 and Loss of Regulatory T Cell Identity at Sites of Inflammation

    doi: 10.1016/j.celrep.2019.01.070

    Figure Lengend Snippet: CNS Treg Cells are Stable and Express Blimp1 in Response to Proinflammatory Cytokines (A) Mononuclear cells were isolated from the CNS of EAE mice at the peak of disease and were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin to analyze the expression of IL-10, IL-17, and IFN-γ in Treg cells by flow cytometry. Mean of eight biological replicates ± SD, derived from three independent experiments. Symbols depict individual mice (bars, mean ± SD). (B) CD4 + Foxp3 + Treg cells were sorted from the CNS and spleen (SPL) of Foxp3 (GFP) reporter mice at the peak of EAE and subjected to RNA-seq. Principal-component analysis. (C) Foxp3 expression by intracellular staining of splenic Treg cells and CNS Treg cells at the peak of EAE. Mean of six biological replicates ± SD, derived from two independent experiments. Symbols depict individual mice (bars, mean ± SD), t test, p

    Article Snippet: Antibodies and flow cytometry Cell suspensions from lymphoid organs were stained with fluorochrome-conjugated anti-mouse CD4 (RM4-5), CD25 (PC61.5), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD62L (MEL-14), CD90.1 (OX-7), GITR (CD357) (DTA-1), Ki67 (16A8), CD126 (D7715A7) and KLRG1 (2F1), which were purchased from either Biolegend, eBioscience or BD Biosciences.

    Techniques: Isolation, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Derivative Assay, RNA Sequencing Assay, Staining

    Lack of Blimp1 in Treg Cells Results in Instability of Treg Cells through Epigenetic Changes (A) Schematic scheme of experimental set up. (B and C) Total Treg cells, namely, both control Foxp3 Treg cells ( Foxp3 Cre ) (B) or wild-type (C) and Blimp1 ΔFoxp3 Tre cells together, were sorted from spleen and draining lymph node of immunized mixed bone marrow chimeras on day 8 post-immunization and were transferred into Rag1 −/− recipient mice along with congenically marked CD25 - CD44 − CD90.1 + naive conventional CD4 + T cells (Thy 1.1 Tconv). On day 1 post-transfer, the recipient mice were immunized with MOG(35-55) and CFA. Mononuclear cells were isolated from the spleen and draining lymph node for analysis 8 days post-immunization. (C) Ki67 staining in wild-type and Blimp1 ΔFoxp3 Treg cells re-isolated from the spleen of the secondary hosts. Representative of three independent biological replicates. (D) Foxp3 expression was analyzed by intracellular staining. Frequency and geomean of Foxp3 + cells within the transferred Foxp3 Cre control Treg cells and Blimp1 ΔFoxp3 Treg cells isolated from the spleen of secondary hosts. Cumulative data of five biological replicates derived from two independent experiments. Symbols depict individual mice (bars, mean ± SD). Student’s t test ( ∗ p

    Journal: Cell Reports

    Article Title: Blimp1 Prevents Methylation of Foxp3 and Loss of Regulatory T Cell Identity at Sites of Inflammation

    doi: 10.1016/j.celrep.2019.01.070

    Figure Lengend Snippet: Lack of Blimp1 in Treg Cells Results in Instability of Treg Cells through Epigenetic Changes (A) Schematic scheme of experimental set up. (B and C) Total Treg cells, namely, both control Foxp3 Treg cells ( Foxp3 Cre ) (B) or wild-type (C) and Blimp1 ΔFoxp3 Tre cells together, were sorted from spleen and draining lymph node of immunized mixed bone marrow chimeras on day 8 post-immunization and were transferred into Rag1 −/− recipient mice along with congenically marked CD25 - CD44 − CD90.1 + naive conventional CD4 + T cells (Thy 1.1 Tconv). On day 1 post-transfer, the recipient mice were immunized with MOG(35-55) and CFA. Mononuclear cells were isolated from the spleen and draining lymph node for analysis 8 days post-immunization. (C) Ki67 staining in wild-type and Blimp1 ΔFoxp3 Treg cells re-isolated from the spleen of the secondary hosts. Representative of three independent biological replicates. (D) Foxp3 expression was analyzed by intracellular staining. Frequency and geomean of Foxp3 + cells within the transferred Foxp3 Cre control Treg cells and Blimp1 ΔFoxp3 Treg cells isolated from the spleen of secondary hosts. Cumulative data of five biological replicates derived from two independent experiments. Symbols depict individual mice (bars, mean ± SD). Student’s t test ( ∗ p

    Article Snippet: Antibodies and flow cytometry Cell suspensions from lymphoid organs were stained with fluorochrome-conjugated anti-mouse CD4 (RM4-5), CD25 (PC61.5), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD62L (MEL-14), CD90.1 (OX-7), GITR (CD357) (DTA-1), Ki67 (16A8), CD126 (D7715A7) and KLRG1 (2F1), which were purchased from either Biolegend, eBioscience or BD Biosciences.

    Techniques: Mouse Assay, Isolation, Staining, Expressing, Derivative Assay