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Histamine dihydrochloride (HDC)-induced differentiation of leukemic cells is NOX2-dependent. (A) <t>FACS-plots</t> showing NOX2 and H 2 R expression on wild-type (WT) and NOX2 -KO <t>PLB-985</t> cells. Expression of CD11b (B,C) , FPR1 (D) , and FPR2 (E) on WT and NOX2 -KO PLB-985 cells cultured in the presence or absence of HDC or dimethyl sulfoxide (DMSO) as determined by flow cytometry. (F) FACS-plot showing NOX2 and H 2 R expression by OCI-AML3 cells. Expression of CD11b (G) , CD14 (H) , FPR1 (I) , and FPR2 (J) on OCI-AML3 cells cultured in the presence or absence of HDC or DMSO. Abbreviations: MFI, median fluorescence intensity. ANOVA; * p
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1) Product Images from "Anti-Leukemic Properties of Histamine in Monocytic Leukemia: The Role of NOX2"

Article Title: Anti-Leukemic Properties of Histamine in Monocytic Leukemia: The Role of NOX2

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2018.00218

Histamine dihydrochloride (HDC)-induced differentiation of leukemic cells is NOX2-dependent. (A) FACS-plots showing NOX2 and H 2 R expression on wild-type (WT) and NOX2 -KO PLB-985 cells. Expression of CD11b (B,C) , FPR1 (D) , and FPR2 (E) on WT and NOX2 -KO PLB-985 cells cultured in the presence or absence of HDC or dimethyl sulfoxide (DMSO) as determined by flow cytometry. (F) FACS-plot showing NOX2 and H 2 R expression by OCI-AML3 cells. Expression of CD11b (G) , CD14 (H) , FPR1 (I) , and FPR2 (J) on OCI-AML3 cells cultured in the presence or absence of HDC or DMSO. Abbreviations: MFI, median fluorescence intensity. ANOVA; * p
Figure Legend Snippet: Histamine dihydrochloride (HDC)-induced differentiation of leukemic cells is NOX2-dependent. (A) FACS-plots showing NOX2 and H 2 R expression on wild-type (WT) and NOX2 -KO PLB-985 cells. Expression of CD11b (B,C) , FPR1 (D) , and FPR2 (E) on WT and NOX2 -KO PLB-985 cells cultured in the presence or absence of HDC or dimethyl sulfoxide (DMSO) as determined by flow cytometry. (F) FACS-plot showing NOX2 and H 2 R expression by OCI-AML3 cells. Expression of CD11b (G) , CD14 (H) , FPR1 (I) , and FPR2 (J) on OCI-AML3 cells cultured in the presence or absence of HDC or DMSO. Abbreviations: MFI, median fluorescence intensity. ANOVA; * p

Techniques Used: FACS, Expressing, Cell Culture, Flow Cytometry, Cytometry, Fluorescence

Histamine dihydrochloride (HDC) facilitates the differentiation of monocytic primary leukemic cells and may be preferentially efficacious in monocytic forms of leukemia. FACS-plots showing live peripheral blood mononuclear cells from representative newly diagnosed patients with (A) FAB-M0 acute myeloid leukemia (AML) with a dominant immature leukemic population (CD34 + CD33 − CD14 − ) and (B) FAB-M4 AML with two distinct populations: an immature blast population (CD34 + CD33 − CD14 − ) and a mature monocytic population (CD34 − CD33 + CD14 + ). The expression of (C) H 2 R, (D) NOX2, (E) FPR1, and (F) FPR2 on primary AML cells [gated as indicated in (A,B) ] and monocytes from healthy donors was determined by flow cytometry. The M5 leukemia is represented by an open circle. One-way ANOVA. (G–I) Median fluorescence intensity as determined by flow cytometry of (G) HLA-DR, (H) FPR1, and (I) FPR2 on live primary monocytic AML cells (FAB: M4/M5) or non-monocytic AML cells (FAB: M0–M2) cultured for 5 days with GM-CSF/IL-4 in the presence or absence of HDC. Wilcoxon matched pair’s test. * p
Figure Legend Snippet: Histamine dihydrochloride (HDC) facilitates the differentiation of monocytic primary leukemic cells and may be preferentially efficacious in monocytic forms of leukemia. FACS-plots showing live peripheral blood mononuclear cells from representative newly diagnosed patients with (A) FAB-M0 acute myeloid leukemia (AML) with a dominant immature leukemic population (CD34 + CD33 − CD14 − ) and (B) FAB-M4 AML with two distinct populations: an immature blast population (CD34 + CD33 − CD14 − ) and a mature monocytic population (CD34 − CD33 + CD14 + ). The expression of (C) H 2 R, (D) NOX2, (E) FPR1, and (F) FPR2 on primary AML cells [gated as indicated in (A,B) ] and monocytes from healthy donors was determined by flow cytometry. The M5 leukemia is represented by an open circle. One-way ANOVA. (G–I) Median fluorescence intensity as determined by flow cytometry of (G) HLA-DR, (H) FPR1, and (I) FPR2 on live primary monocytic AML cells (FAB: M4/M5) or non-monocytic AML cells (FAB: M0–M2) cultured for 5 days with GM-CSF/IL-4 in the presence or absence of HDC. Wilcoxon matched pair’s test. * p

Techniques Used: FACS, Expressing, Flow Cytometry, Cytometry, Fluorescence, Cell Culture

2) Product Images from "The eicosanoids leukotriene D4 and prostaglandin E2 promote the tumorigenicity of colon cancer-initiating cells in a xenograft mouse model"

Article Title: The eicosanoids leukotriene D4 and prostaglandin E2 promote the tumorigenicity of colon cancer-initiating cells in a xenograft mouse model

Journal: BMC Cancer

doi: 10.1186/s12885-016-2466-z

FACS analysis and immunohistochemistry of xenograft ALDH + HCT-116 cell tumors treated with LTD 4 or PGE 2 . a Representative dot plots and corresponding graphs of FACS analysis of the percentage of ALDH + cells in dissociated tumors from vehicle (ethanol), LTD 4 or PGE 2 treated groups respectively. b – e Immunohistochemical analysis of ALDH, Dclk1, β-catenin, and COX-2 protein expression levels. The tumors from vehicle- (ethanol), LTD 4 - and PGE 2 -treated mice were processed for immunohistochemical analysis. Representative images (40×) and corresponding bar graphs show staining scores of ( b ) ALDH, ( c ) Dclk1, ( d ) β-catenin, and ( e ) COX-2 proteins in tumors. The final scores represent the sum of the staining intensity and staining percentage within tumor areas. The data are expressed as means ± SEM, n = 6 mice in each group. * P
Figure Legend Snippet: FACS analysis and immunohistochemistry of xenograft ALDH + HCT-116 cell tumors treated with LTD 4 or PGE 2 . a Representative dot plots and corresponding graphs of FACS analysis of the percentage of ALDH + cells in dissociated tumors from vehicle (ethanol), LTD 4 or PGE 2 treated groups respectively. b – e Immunohistochemical analysis of ALDH, Dclk1, β-catenin, and COX-2 protein expression levels. The tumors from vehicle- (ethanol), LTD 4 - and PGE 2 -treated mice were processed for immunohistochemical analysis. Representative images (40×) and corresponding bar graphs show staining scores of ( b ) ALDH, ( c ) Dclk1, ( d ) β-catenin, and ( e ) COX-2 proteins in tumors. The final scores represent the sum of the staining intensity and staining percentage within tumor areas. The data are expressed as means ± SEM, n = 6 mice in each group. * P

Techniques Used: FACS, Immunohistochemistry, Expressing, Mouse Assay, Staining

FACS analysis of xenograft ALDH + HCT-116 cell tumors treated with LTD 4 or PGE 2 . a – c Representative dot plots and corresponding graphs of FACS analysis of the percentage of CD45 + cells in dissociated tumors from vehicle (ethanol), LTD 4 or PGE 2 treated groups respectively. a CD45 + , ( b ) LY6G, and ( c ) CD4 + cells in dissociated tumors from vehicle (ethanol), LTD 4 or PGE 2 treated groups respectively. The data are expressed as means ± SEM, n = 6 mice in each group. * P
Figure Legend Snippet: FACS analysis of xenograft ALDH + HCT-116 cell tumors treated with LTD 4 or PGE 2 . a – c Representative dot plots and corresponding graphs of FACS analysis of the percentage of CD45 + cells in dissociated tumors from vehicle (ethanol), LTD 4 or PGE 2 treated groups respectively. a CD45 + , ( b ) LY6G, and ( c ) CD4 + cells in dissociated tumors from vehicle (ethanol), LTD 4 or PGE 2 treated groups respectively. The data are expressed as means ± SEM, n = 6 mice in each group. * P

Techniques Used: FACS, Mouse Assay

3) Product Images from "Limiting the priming dose of a SARS CoV-2 vaccine improves virus-specific immunity"

Article Title: Limiting the priming dose of a SARS CoV-2 vaccine improves virus-specific immunity

Journal: bioRxiv

doi: 10.1101/2021.03.31.437931

A LD/SD vaccine regimen elicits superior CD8 T cells compared to a SD/SD vaccine regimen. (A) Experimental approach for evaluating how the priming dose of an Ad5-SARS-2 spike vaccine affects CD8 T cell responses in C57BL/6 mice. (B) Representative FACS plots showing the frequencies of SARS CoV-2-specific CD8 T cells (K b VL8+) in PBMCs. (C) Summary of SARS CoV-2-specific CD8 T cell responses in PBMCs. (D) Representative FACS plots showing the frequencies of SARS CoV-2-specific CD8 T cells (K b VL8+) in tissues. (E) Summary of SARS CoV-2-specific CD8 T cell responses in tissues. Data are from one experiment with n=5 per group. Experiment was repeated two additional times with similar results. Indicated P values were determined by Mann-Whitney U test. Error bars represent SEM.
Figure Legend Snippet: A LD/SD vaccine regimen elicits superior CD8 T cells compared to a SD/SD vaccine regimen. (A) Experimental approach for evaluating how the priming dose of an Ad5-SARS-2 spike vaccine affects CD8 T cell responses in C57BL/6 mice. (B) Representative FACS plots showing the frequencies of SARS CoV-2-specific CD8 T cells (K b VL8+) in PBMCs. (C) Summary of SARS CoV-2-specific CD8 T cell responses in PBMCs. (D) Representative FACS plots showing the frequencies of SARS CoV-2-specific CD8 T cells (K b VL8+) in tissues. (E) Summary of SARS CoV-2-specific CD8 T cell responses in tissues. Data are from one experiment with n=5 per group. Experiment was repeated two additional times with similar results. Indicated P values were determined by Mann-Whitney U test. Error bars represent SEM.

Techniques Used: Mouse Assay, FACS, MANN-WHITNEY

Single cell TCR-seq analyses demonstrate that the prime dose does not alter public TCR clonotypes. Mice were primed with a LD (10 6 PFU) or a SD (10 9 PFU) of Ad5-SARS-2 spike, and at day 28, splenic CD8 T cells were MACS-sorted. Subsequently, live, CD8+, CD44+, K b VL8+ cells were FACS-sorted to ~99% purity for scTCR-Seq. Pie chart showing the distribution of TCRa and TCRb gene usage after SD prime (A) and LD prime (B). Total number above the pie chart show the number of single cells selected for the analyses, and different colors highlight the top 5 TCR usages and their relative proportion in each population.
Figure Legend Snippet: Single cell TCR-seq analyses demonstrate that the prime dose does not alter public TCR clonotypes. Mice were primed with a LD (10 6 PFU) or a SD (10 9 PFU) of Ad5-SARS-2 spike, and at day 28, splenic CD8 T cells were MACS-sorted. Subsequently, live, CD8+, CD44+, K b VL8+ cells were FACS-sorted to ~99% purity for scTCR-Seq. Pie chart showing the distribution of TCRa and TCRb gene usage after SD prime (A) and LD prime (B). Total number above the pie chart show the number of single cells selected for the analyses, and different colors highlight the top 5 TCR usages and their relative proportion in each population.

Techniques Used: Mouse Assay, Magnetic Cell Separation, FACS

Phenotypic validation of CD8 T cell responses after a single prime with the Ad5-SARS-2 spike vaccine. (A) Representative FACS plots showing the frequencies of SARS CoV-2-specific CD8 T cells (K b VL8) that differentiate into effector memory and central memory T cell subsets. (B) Summary of effector memory and central memory T cell subsets. (C) CD127 expression. (D) CD62L expression. (E) CD44 expression. (F) PD-1 expression. Panels B-F are gated from splenic SARS CoV-2-specific CD8 T cells (K b VL8). All data are from day 28 post-prime. Data are from one experiment with n=5 per group. Experiment was repeated two additional times with similar results. Indicated P values were determined by Mann-Whitney U test.
Figure Legend Snippet: Phenotypic validation of CD8 T cell responses after a single prime with the Ad5-SARS-2 spike vaccine. (A) Representative FACS plots showing the frequencies of SARS CoV-2-specific CD8 T cells (K b VL8) that differentiate into effector memory and central memory T cell subsets. (B) Summary of effector memory and central memory T cell subsets. (C) CD127 expression. (D) CD62L expression. (E) CD44 expression. (F) PD-1 expression. Panels B-F are gated from splenic SARS CoV-2-specific CD8 T cells (K b VL8). All data are from day 28 post-prime. Data are from one experiment with n=5 per group. Experiment was repeated two additional times with similar results. Indicated P values were determined by Mann-Whitney U test.

Techniques Used: FACS, Expressing, MANN-WHITNEY

Single cell RNA-seq analyses demonstrate that a low dose prime favors central memory CD8 T cell differentiation. Mice were immunized with 10 6 or 10 9 PFU of Ad5-SARS-2 spike, and at day 28, splenic CD8 T cells were MACS-sorted. Subsequently, live, CD8+, CD44+, K b VL8+ cells were FACS-sorted to ~99% purity for scRNA-seq. (A) UMAP plots showing populations colored by regimen (left plot). Standard and low dose cells were cluster separately and UMAP (right plot) shows unsupervised cell clusters. (B) Heatmap showing row-standardized expression of selected effector and memory genes (middle rows) or gene signatures (bottom rows). For each population, percentages of cells in each cluster are indicated (top row). (C) Violin plot showing the normalized expression of the Terminal Effector signature in the Standard and Low dose populations.
Figure Legend Snippet: Single cell RNA-seq analyses demonstrate that a low dose prime favors central memory CD8 T cell differentiation. Mice were immunized with 10 6 or 10 9 PFU of Ad5-SARS-2 spike, and at day 28, splenic CD8 T cells were MACS-sorted. Subsequently, live, CD8+, CD44+, K b VL8+ cells were FACS-sorted to ~99% purity for scRNA-seq. (A) UMAP plots showing populations colored by regimen (left plot). Standard and low dose cells were cluster separately and UMAP (right plot) shows unsupervised cell clusters. (B) Heatmap showing row-standardized expression of selected effector and memory genes (middle rows) or gene signatures (bottom rows). For each population, percentages of cells in each cluster are indicated (top row). (C) Violin plot showing the normalized expression of the Terminal Effector signature in the Standard and Low dose populations.

Techniques Used: RNA Sequencing Assay, Cell Differentiation, Mouse Assay, Magnetic Cell Separation, FACS, Expressing

A LD/SD vaccine regimen elicits more functional CD8 T cell responses compared to a SD/SD vaccine regimen. In panels A-D, splenocytes were incubated with overlapping SARS CoV-2 peptide pools for 5 hr at 37°C in the presence of GolgiStop and GolgiPlug. (A) Representative FACS plots showing the frequencies of cytokine expressing SARS CoV-2-specific CD8 T cells. (B) Summary of SARS CoV-2-specific CD8 T cells that express the degranulation marker CD107a. (C) Summary of SARS CoV-2-specific CD8 T cells that express IFNγ. (D) Summary of SARS CoV-2-specific CD4 T cells that express IFNγ. (E) Representative FACS plots showing the frequencies of granzyme B and Ki67 expressing CD8 T cells. (F) Summary of Ki67 expression. (G) Summary of granzyme B expression. Panels E-G are gated on K b VL8+ cells (SARS CoV-2-specific). Data from panels F-G are indicated as mean fluorescence intensity (MFI). All data are from spleen. Data are from one experiment with n=4–5 per group. Experiment was repeated once with similar results. Indicated P values were determined by Mann-Whitney U test.
Figure Legend Snippet: A LD/SD vaccine regimen elicits more functional CD8 T cell responses compared to a SD/SD vaccine regimen. In panels A-D, splenocytes were incubated with overlapping SARS CoV-2 peptide pools for 5 hr at 37°C in the presence of GolgiStop and GolgiPlug. (A) Representative FACS plots showing the frequencies of cytokine expressing SARS CoV-2-specific CD8 T cells. (B) Summary of SARS CoV-2-specific CD8 T cells that express the degranulation marker CD107a. (C) Summary of SARS CoV-2-specific CD8 T cells that express IFNγ. (D) Summary of SARS CoV-2-specific CD4 T cells that express IFNγ. (E) Representative FACS plots showing the frequencies of granzyme B and Ki67 expressing CD8 T cells. (F) Summary of Ki67 expression. (G) Summary of granzyme B expression. Panels E-G are gated on K b VL8+ cells (SARS CoV-2-specific). Data from panels F-G are indicated as mean fluorescence intensity (MFI). All data are from spleen. Data are from one experiment with n=4–5 per group. Experiment was repeated once with similar results. Indicated P values were determined by Mann-Whitney U test.

Techniques Used: Functional Assay, Incubation, FACS, Expressing, Marker, Fluorescence, MANN-WHITNEY

A LD prime elicits CD8 T cell responses with intrinsically superior anamnestic capacity. CD45.2+ mice were immunized intramuscularly with 10 6 or 10 9 PFU of Ad5-SARS-2 spike, and at day 28, splenic CD8 T cells were MACS-sorted. Subsequently, live, CD8+, CD44+, K b VL8+ cells were FACS-sorted to ~99% purity, and numbers were normalized for adoptive transfer into CD45.1+ recipient mice. (A) Experimental approach for evaluating secondary expansion of donor CD8 T cells. (B) Representative FACS plots showing the frequencies of donor CD8 T cells after boosting. (C) Summary of donor-derived CD8 T cells in PBMCs. (D) Summary of donor-derived CD8 T cells in spleen. (E) Summary of donor-derived CD8 T cells in draining lymph nodes. Data from panels B-E are from day 14 post-boost. Data are from one experiment with n=5 per group. Experiment was repeated once with similar results. Indicated P values were determined by Mann-Whitney U test.
Figure Legend Snippet: A LD prime elicits CD8 T cell responses with intrinsically superior anamnestic capacity. CD45.2+ mice were immunized intramuscularly with 10 6 or 10 9 PFU of Ad5-SARS-2 spike, and at day 28, splenic CD8 T cells were MACS-sorted. Subsequently, live, CD8+, CD44+, K b VL8+ cells were FACS-sorted to ~99% purity, and numbers were normalized for adoptive transfer into CD45.1+ recipient mice. (A) Experimental approach for evaluating secondary expansion of donor CD8 T cells. (B) Representative FACS plots showing the frequencies of donor CD8 T cells after boosting. (C) Summary of donor-derived CD8 T cells in PBMCs. (D) Summary of donor-derived CD8 T cells in spleen. (E) Summary of donor-derived CD8 T cells in draining lymph nodes. Data from panels B-E are from day 14 post-boost. Data are from one experiment with n=5 per group. Experiment was repeated once with similar results. Indicated P values were determined by Mann-Whitney U test.

Techniques Used: Mouse Assay, Magnetic Cell Separation, FACS, Adoptive Transfer Assay, Derivative Assay, MANN-WHITNEY

4) Product Images from "Cell-Intrinsic Control of Interneuron Migration Drives Cortical Morphogenesis"

Article Title: Cell-Intrinsic Control of Interneuron Migration Drives Cortical Morphogenesis

Journal: Cell

doi: 10.1016/j.cell.2018.01.031

Assessment of Migration Parameters in Cortical Interneurons Lacking Ccp1 Expression, Related to Figure 1 (A) Schematic representation of the targeting vector used and all possible alleles for Ccp1 gene. Orange bar: genomic DNA. Black boxes: exons with their corresponding number. Green and purple arrowheads: LoxP and Flp sequences, respectively. White bar: neo cassette, with the neomycin resistance gene (white box). Blue lines: zone of sequence homology for homologous recombination, with the corresponding size in kbp. Black arrowheads: primers used for the PCR genotyping. (B) Normalized expression levels of CCP and TTLL mRNAs from extracted RNA of FACS E13.5 CCP1 WT and CCP1 cKO GEs. Values are expressed as FPKM, n = 3 embryos from independent female donors. (C–F) Time-lapse experiments of CCP1 WT and CCP1 cKO cINs from explant co-cultures. Quantification of growth cone splitting (emergence of a secondary branch from the growth cone) (C), average number of different neurite types on cINs during migration (I = primary neurites, II = secondary and III = tertiary neurites) (D), neuritic length (E) and frequency of nucleokinesis (F), respectively, n = 26-52 cells from at least 3 independent cultures, two-way ANOVA. (G and H) Representative examples of CCP1 WT and CCP1 cKO nucleus (G) and centroid displacements (H) measured by time-lapse acquisition in E13.5 GEs explants. The traveled distance between two time points is plotted and every displacement above 5μm (blue dotted line) is considered as a nucleokinesis. (I) Quantification of the total time of nuclear movement in CCP1 WT and CCP1 cKO measured in time-lapse acquisition of E13.5 organotypic slice culture. (J–L) Time-lapse analysis of cINs in explant co-culture showing average speed (J), pausing time (K), and directionality (L) after overexpression of Ccp1 (CCP1 OE), its catalytic dead form (CCP1dead OE), or a control (Tomato OE), n = 22-78 cells from at least 3 independent cultures, one-way ANOVA. All graphs contain bars representing SEM.
Figure Legend Snippet: Assessment of Migration Parameters in Cortical Interneurons Lacking Ccp1 Expression, Related to Figure 1 (A) Schematic representation of the targeting vector used and all possible alleles for Ccp1 gene. Orange bar: genomic DNA. Black boxes: exons with their corresponding number. Green and purple arrowheads: LoxP and Flp sequences, respectively. White bar: neo cassette, with the neomycin resistance gene (white box). Blue lines: zone of sequence homology for homologous recombination, with the corresponding size in kbp. Black arrowheads: primers used for the PCR genotyping. (B) Normalized expression levels of CCP and TTLL mRNAs from extracted RNA of FACS E13.5 CCP1 WT and CCP1 cKO GEs. Values are expressed as FPKM, n = 3 embryos from independent female donors. (C–F) Time-lapse experiments of CCP1 WT and CCP1 cKO cINs from explant co-cultures. Quantification of growth cone splitting (emergence of a secondary branch from the growth cone) (C), average number of different neurite types on cINs during migration (I = primary neurites, II = secondary and III = tertiary neurites) (D), neuritic length (E) and frequency of nucleokinesis (F), respectively, n = 26-52 cells from at least 3 independent cultures, two-way ANOVA. (G and H) Representative examples of CCP1 WT and CCP1 cKO nucleus (G) and centroid displacements (H) measured by time-lapse acquisition in E13.5 GEs explants. The traveled distance between two time points is plotted and every displacement above 5μm (blue dotted line) is considered as a nucleokinesis. (I) Quantification of the total time of nuclear movement in CCP1 WT and CCP1 cKO measured in time-lapse acquisition of E13.5 organotypic slice culture. (J–L) Time-lapse analysis of cINs in explant co-culture showing average speed (J), pausing time (K), and directionality (L) after overexpression of Ccp1 (CCP1 OE), its catalytic dead form (CCP1dead OE), or a control (Tomato OE), n = 22-78 cells from at least 3 independent cultures, one-way ANOVA. All graphs contain bars representing SEM.

Techniques Used: Migration, Expressing, Plasmid Preparation, Sequencing, Homologous Recombination, Polymerase Chain Reaction, FACS, Co-Culture Assay, Over Expression

CCP1 Promotes the Saltatory Migration of cINs (A) Immunodetection of polyglutamate side chains (GT335 antibody) on cINs explants. cINs express Cre-GFP and nuclei are counterstained with DAPI. The white arrow points the leading process. Scale bar, 10 μm. (B) Normalized expression levels of CCP and TTLL mRNAs in E13.5 WT cINs, n = 3 embryos per group from 3 females. (C) ISH of Ccp1 on a coronal section of E13.5. (D) Subcellular distribution of CCP1 (red) in WT migrating cINs. Scale bars, 5 μm (top), 2 μm (bottom). (E) Normalized expression levels of Ccp1 exons 20 and 21 in FACS-purified cINs from 3 E13.5 CCP1 WT or cKO mouse embryos. (F) Glutamylation levels (PolyE or GT335 antibodies) on tubulin extracted from GEs of CCP1 WT and cKO embryos at E13.5. (G) PolyE and GT335 immunoreactivity normalized on tubulin and actin and expressed as percentage of control, n = 5–7 embryos per group, non-parametric t test, p
Figure Legend Snippet: CCP1 Promotes the Saltatory Migration of cINs (A) Immunodetection of polyglutamate side chains (GT335 antibody) on cINs explants. cINs express Cre-GFP and nuclei are counterstained with DAPI. The white arrow points the leading process. Scale bar, 10 μm. (B) Normalized expression levels of CCP and TTLL mRNAs in E13.5 WT cINs, n = 3 embryos per group from 3 females. (C) ISH of Ccp1 on a coronal section of E13.5. (D) Subcellular distribution of CCP1 (red) in WT migrating cINs. Scale bars, 5 μm (top), 2 μm (bottom). (E) Normalized expression levels of Ccp1 exons 20 and 21 in FACS-purified cINs from 3 E13.5 CCP1 WT or cKO mouse embryos. (F) Glutamylation levels (PolyE or GT335 antibodies) on tubulin extracted from GEs of CCP1 WT and cKO embryos at E13.5. (G) PolyE and GT335 immunoreactivity normalized on tubulin and actin and expressed as percentage of control, n = 5–7 embryos per group, non-parametric t test, p

Techniques Used: Migration, Immunodetection, Expressing, In Situ Hybridization, FACS, Purification

5) Product Images from "RORγt+ cells selectively express redundant cation channels linked to the Golgi apparatus"

Article Title: RORγt+ cells selectively express redundant cation channels linked to the Golgi apparatus

Journal: Scientific Reports

doi: 10.1038/srep23682

Tmem176a and b mRNA expression in intestinal ILC3s. ( a ) Lymphocytes from intestinal lamina propria of RORγt-fate map mice ( Rorc ( γt )- Cre TG × Rosa26-tdRFP ) were isolated. Lineage-negative (CD11b − CD11c − CD19 − TCRαβ − TCRγδ − ) RFP + NK1.1 − ILC3-enriched and RFP − NK1.1 + ILC1s were FACS-sorted. In parallel, CD11b/c + and lineage-negative NK1.1 + conventional NK/ILC1s were FACS-sorted from the spleen. ( b ) Expression of indicated genes (mean ± SD) was assessed by quantitative RT-PCR in each population isolated from 3–4 independent mice. Statistically significant differences between ILC3s and the other populations are indicated: *p
Figure Legend Snippet: Tmem176a and b mRNA expression in intestinal ILC3s. ( a ) Lymphocytes from intestinal lamina propria of RORγt-fate map mice ( Rorc ( γt )- Cre TG × Rosa26-tdRFP ) were isolated. Lineage-negative (CD11b − CD11c − CD19 − TCRαβ − TCRγδ − ) RFP + NK1.1 − ILC3-enriched and RFP − NK1.1 + ILC1s were FACS-sorted. In parallel, CD11b/c + and lineage-negative NK1.1 + conventional NK/ILC1s were FACS-sorted from the spleen. ( b ) Expression of indicated genes (mean ± SD) was assessed by quantitative RT-PCR in each population isolated from 3–4 independent mice. Statistically significant differences between ILC3s and the other populations are indicated: *p

Techniques Used: Expressing, Mouse Assay, Isolation, FACS, Quantitative RT-PCR

Tmem176b single-deficient mouse susceptibility to the development of psoriasis-like dermatitis. ( a ) Psoriasis-like dermatitis was induced in WT mice by topical application of imiquimod (IMQ) cream on the shaved back skin. At day 4, draining (inguinal) lymph nodes were harvested and TCRγδ + and TCRβ + CD4 + T cells were FACS-sorted. ( b ) Expression of indicated genes was assessed by quantitative RT-PCR. Each dot represents an individual mouse (n = 6 in each group). Statistically significant differences are indicated: **p
Figure Legend Snippet: Tmem176b single-deficient mouse susceptibility to the development of psoriasis-like dermatitis. ( a ) Psoriasis-like dermatitis was induced in WT mice by topical application of imiquimod (IMQ) cream on the shaved back skin. At day 4, draining (inguinal) lymph nodes were harvested and TCRγδ + and TCRβ + CD4 + T cells were FACS-sorted. ( b ) Expression of indicated genes was assessed by quantitative RT-PCR. Each dot represents an individual mouse (n = 6 in each group). Statistically significant differences are indicated: **p

Techniques Used: Mouse Assay, FACS, Expressing, Quantitative RT-PCR

6) Product Images from "Neural precursor cell–secreted TGF- β2 redirects inflammatory monocyte-derived cells in CNS autoimmunity"

Article Title: Neural precursor cell–secreted TGF- β2 redirects inflammatory monocyte-derived cells in CNS autoimmunity

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI92387

NPC treatment alters the gene expression signature of CNS-infiltrating inflammatory MCs in EAE. ( A ) Cohorts of 4–7 MOG 35–55 -immunized C57BL/6 mice intrathecally treated with either PBS or NPCs at the peak of the disease (2–4 days after clinical onset). At 7 days after transplantation, CNS tissues were pooled and CNS-infiltrating MCs were FACS-sorted according to the phenotype CD45 hi Ly6G – CD11b + Ly6C hi MHC-II + . Sorting strategy used for 3 independent FACS sorting experiments is shown. ( B ) Next-generation sequencing was performed on RNA extracted from sorted cells of 3 independent experiments and respective CNS harvests. Six hundred ten genes that are significantly altered in 3 different statistical tests to a minimum significance threshold of P ≤ 0.01 are shown in the heatmap. ( C ) Volcano plot showing the fold change and significance of genes in MCs from PBS- versus NPC-treated EAE mice. ( D ). Black and gray nodes represent enriched pathways with sizes corresponding to FDR-adjusted enrichment P value ( P ≤ 0.05). Red dots represent upregulated genes and blue dots downregulated genes, whereas the dot size indicates significance ( P ≤ 0.01).
Figure Legend Snippet: NPC treatment alters the gene expression signature of CNS-infiltrating inflammatory MCs in EAE. ( A ) Cohorts of 4–7 MOG 35–55 -immunized C57BL/6 mice intrathecally treated with either PBS or NPCs at the peak of the disease (2–4 days after clinical onset). At 7 days after transplantation, CNS tissues were pooled and CNS-infiltrating MCs were FACS-sorted according to the phenotype CD45 hi Ly6G – CD11b + Ly6C hi MHC-II + . Sorting strategy used for 3 independent FACS sorting experiments is shown. ( B ) Next-generation sequencing was performed on RNA extracted from sorted cells of 3 independent experiments and respective CNS harvests. Six hundred ten genes that are significantly altered in 3 different statistical tests to a minimum significance threshold of P ≤ 0.01 are shown in the heatmap. ( C ) Volcano plot showing the fold change and significance of genes in MCs from PBS- versus NPC-treated EAE mice. ( D ). Black and gray nodes represent enriched pathways with sizes corresponding to FDR-adjusted enrichment P value ( P ≤ 0.05). Red dots represent upregulated genes and blue dots downregulated genes, whereas the dot size indicates significance ( P ≤ 0.01).

Techniques Used: Expressing, Mouse Assay, Transplantation Assay, FACS, Next-Generation Sequencing

7) Product Images from "T CELL SPECIFIC ADAPTOR PROTEIN REGULATES MITOCHONDRIAL FUNCTION AND CD4+ T REGULATORY CELL ACTIVITY IN VIVO FOLLOWING TRANSPLANTATION"

Article Title: T CELL SPECIFIC ADAPTOR PROTEIN REGULATES MITOCHONDRIAL FUNCTION AND CD4+ T REGULATORY CELL ACTIVITY IN VIVO FOLLOWING TRANSPLANTATION

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

doi: 10.4049/jimmunol.1801604

TSAd knockout recipients are resistant to the graft prolonging effects of costimulatory blockade. Fully MHC mismatched BALB/c hearts were transplanted into WT or TSAd knockout (ΔTSAd) recipients respectively and were treated with (A) anti-CD40L or (B) CTLA4-Ig intraperitoneally on days 0, 2 and 4 post-transplantation. Graft survival was monitored by palpation. Statistics were performed by comparing outcomes in treated ΔTSAd vs. treated WT recipients. (C) Fully MHC mismatched BALB/c hearts were transplanted into C57BL/6 Rag2 Il2rg double knock-out recipients. On day 2 post-transplantation, recipients received FACS-sorted CD4 + Foxp3 + Treg cells (2×10 5 ) from either WT or TSAd knockout mice by tail vein injection. On day 18 post-transplantation, recipients were challenged with WT CD4 + CD25 − Teffs (3×10 6 cells) by tail vein injection. Control recipients did not receive Tregs on day 2. Graft survival following Teff transfer was evaluated by palpation. Statistics in A-C were performed using the Gehan-Breslow-Wilcoxon test.
Figure Legend Snippet: TSAd knockout recipients are resistant to the graft prolonging effects of costimulatory blockade. Fully MHC mismatched BALB/c hearts were transplanted into WT or TSAd knockout (ΔTSAd) recipients respectively and were treated with (A) anti-CD40L or (B) CTLA4-Ig intraperitoneally on days 0, 2 and 4 post-transplantation. Graft survival was monitored by palpation. Statistics were performed by comparing outcomes in treated ΔTSAd vs. treated WT recipients. (C) Fully MHC mismatched BALB/c hearts were transplanted into C57BL/6 Rag2 Il2rg double knock-out recipients. On day 2 post-transplantation, recipients received FACS-sorted CD4 + Foxp3 + Treg cells (2×10 5 ) from either WT or TSAd knockout mice by tail vein injection. On day 18 post-transplantation, recipients were challenged with WT CD4 + CD25 − Teffs (3×10 6 cells) by tail vein injection. Control recipients did not receive Tregs on day 2. Graft survival following Teff transfer was evaluated by palpation. Statistics in A-C were performed using the Gehan-Breslow-Wilcoxon test.

Techniques Used: Knock-Out, Transplantation Assay, FACS, Mouse Assay, Injection

8) Product Images from "TLR4 signaling improves PD-1 blockade therapy during chronic viral infection"

Article Title: TLR4 signaling improves PD-1 blockade therapy during chronic viral infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007583

Effects of LPS on CD8 T cells in chronically infected mice. ( A ) Experimental outline for evaluating the effect of LPS on total activated CD8 T cells or virus-specific CD8 T cells during a chronic viral infection. Mice chronically infected with LCMV Cl-13 received PD-L1 blocking antibodies combined with LPS. ( B ) Summary of total activated CD8 T cells in blood. ( C ) Summary of virus-specific CD8 T cells in blood. ( D ) Representative FACS plots showing the frequencies of virus-specific (D b GP276+) CD8 T cells in blood and tissues. ( E ) Summary of virus-specific (D b GP276+) CD8 T cells in spleen. ( F ) Summary of virus-specific (D b GP276+) CD8 T cells in liver. ( G ) Summary of virus-specific (D b GP276+) CD8 T cells in lung. ( H ) Summary of virus-specific (D b GP33+) CD8 T cells in spleen. Data are pooled from different experiments; PBMC data are from experiments that were performed 3 times, n = 3–5 mice per experiment; Tissue data are from experiments that were performed 2 times, n = 3–5 mice per experiment; ns, not significant. Indicated p-values for panels B-C compare pre- and post-treatment values for each group using Wilcoxon matched-pairs signed rank test. All other data were analyzed using ANOVA for multiple comparisons with Holm-Sidak’s correction. Error bars represent SEM.
Figure Legend Snippet: Effects of LPS on CD8 T cells in chronically infected mice. ( A ) Experimental outline for evaluating the effect of LPS on total activated CD8 T cells or virus-specific CD8 T cells during a chronic viral infection. Mice chronically infected with LCMV Cl-13 received PD-L1 blocking antibodies combined with LPS. ( B ) Summary of total activated CD8 T cells in blood. ( C ) Summary of virus-specific CD8 T cells in blood. ( D ) Representative FACS plots showing the frequencies of virus-specific (D b GP276+) CD8 T cells in blood and tissues. ( E ) Summary of virus-specific (D b GP276+) CD8 T cells in spleen. ( F ) Summary of virus-specific (D b GP276+) CD8 T cells in liver. ( G ) Summary of virus-specific (D b GP276+) CD8 T cells in lung. ( H ) Summary of virus-specific (D b GP33+) CD8 T cells in spleen. Data are pooled from different experiments; PBMC data are from experiments that were performed 3 times, n = 3–5 mice per experiment; Tissue data are from experiments that were performed 2 times, n = 3–5 mice per experiment; ns, not significant. Indicated p-values for panels B-C compare pre- and post-treatment values for each group using Wilcoxon matched-pairs signed rank test. All other data were analyzed using ANOVA for multiple comparisons with Holm-Sidak’s correction. Error bars represent SEM.

Techniques Used: Infection, Mouse Assay, Blocking Assay, FACS

Gene expression profiling of virus-specific CD8 T cells shows enrichment in IFN-I and CD28 driven genes. ( A ) Experimental outline for comparing the transcriptional signature of virus-specific CD8 T cells. RNA-Seq was performed on FACS-sorted D b GP276+ CD8 T cells from spleen. ( B ) Cell purity following FACS-sorting of virus-specific CD8 T cells. ( C ) PCA comparing the transcriptional landscape of rescued CD8 T cells following PD-L1 blockade alone or combined LPS and PD-L1 blockade. ( D ) Heat map of the most differentially expressed genes between single and combined treatment. ( E ) GSEA plots demonstrating enrichment for IFN-I signaling genes in virus-specific CD8 T cells following combined therapy. ( F ) Radar plots showing expression of various IFN-I driven genes. ( G ) GSEA plots demonstrating enrichment for CD28 costimulation genes in virus-specific CD8 T cells following combined therapy. In panels E and G, DN and UP mean downregulated or upregulated, respectively, relative to previously identified transcriptional signatures used as reference. ( H ) Radar plots showing expression of various CD28 driven genes. The genes selected were shown to be enriched following CD28 costimulation in a prior publication [ 71 ]. Presented data are from one experiment, control (n = 3), PD-L1 therapy alone (n = 3), or combined LPS and PD-L1 therapy (n = 4) at day 15 post-treatment.
Figure Legend Snippet: Gene expression profiling of virus-specific CD8 T cells shows enrichment in IFN-I and CD28 driven genes. ( A ) Experimental outline for comparing the transcriptional signature of virus-specific CD8 T cells. RNA-Seq was performed on FACS-sorted D b GP276+ CD8 T cells from spleen. ( B ) Cell purity following FACS-sorting of virus-specific CD8 T cells. ( C ) PCA comparing the transcriptional landscape of rescued CD8 T cells following PD-L1 blockade alone or combined LPS and PD-L1 blockade. ( D ) Heat map of the most differentially expressed genes between single and combined treatment. ( E ) GSEA plots demonstrating enrichment for IFN-I signaling genes in virus-specific CD8 T cells following combined therapy. ( F ) Radar plots showing expression of various IFN-I driven genes. ( G ) GSEA plots demonstrating enrichment for CD28 costimulation genes in virus-specific CD8 T cells following combined therapy. In panels E and G, DN and UP mean downregulated or upregulated, respectively, relative to previously identified transcriptional signatures used as reference. ( H ) Radar plots showing expression of various CD28 driven genes. The genes selected were shown to be enriched following CD28 costimulation in a prior publication [ 71 ]. Presented data are from one experiment, control (n = 3), PD-L1 therapy alone (n = 3), or combined LPS and PD-L1 therapy (n = 4) at day 15 post-treatment.

Techniques Used: Expressing, RNA Sequencing Assay, FACS

Potent synergism between LPS and PD-1 therapy. ( A ) Absolute numbers of virus-specific CD8 T cells in spleen producing IFN-γ after 5-hr stimulation with LCMV peptides (0.1 μg/mL) in the presence of brefeldin A and monensin at 37°C in 5% CO 2. B) Granzyme B expression in virus-specific (D b GP276+) CD8 T cells from spleen. ( C ) Ki67 expression in virus-specific CD8 T cells from spleen. ( D ) Apoptotic (Annexin+ Live/Dead+) virus-specific CD8 T cells in spleen. ( E ) Representative FACS plots depicting apoptotic virus-specific CD8 T cells in spleen. ( F ) Summary of viral control in sera. Fold-change is calculated by dividing the pre-treatment levels by the post-treatment levels. ( G ) Summary of viral control in spleen (day 15 post-treatment). ( H ) Summary of viral control in lung (day 15 post-treatment). ( I ) Summary of long-term viral control in sera. Virologic control is maintained long-term, but complete viral elimination is not observed. Experimental layout was similar as the one depicted in Fig 1A . For all plaque assays the limit of detection is indicated by a dashed line. Data are pooled from different experiments. Experiments were performed twice, n = 3–5 mice per experiment, except for panel F that included 3 experiments, n = 3–5 mice per experiment; ns, not significant. Statistical analyses for panel A were performed with Kruskal-Wallis test with Dunn’s multiple comparison test; for panels B-D and G-H ANOVA for multiple comparisons with Holm-Sidak’s correction was used; for panels F and I, Wilcoxon matched-pairs signed rank test was used comparing pre-treatment viremia versus day 30 viremia within the same treatment group. Error bars represent SEM.
Figure Legend Snippet: Potent synergism between LPS and PD-1 therapy. ( A ) Absolute numbers of virus-specific CD8 T cells in spleen producing IFN-γ after 5-hr stimulation with LCMV peptides (0.1 μg/mL) in the presence of brefeldin A and monensin at 37°C in 5% CO 2. B) Granzyme B expression in virus-specific (D b GP276+) CD8 T cells from spleen. ( C ) Ki67 expression in virus-specific CD8 T cells from spleen. ( D ) Apoptotic (Annexin+ Live/Dead+) virus-specific CD8 T cells in spleen. ( E ) Representative FACS plots depicting apoptotic virus-specific CD8 T cells in spleen. ( F ) Summary of viral control in sera. Fold-change is calculated by dividing the pre-treatment levels by the post-treatment levels. ( G ) Summary of viral control in spleen (day 15 post-treatment). ( H ) Summary of viral control in lung (day 15 post-treatment). ( I ) Summary of long-term viral control in sera. Virologic control is maintained long-term, but complete viral elimination is not observed. Experimental layout was similar as the one depicted in Fig 1A . For all plaque assays the limit of detection is indicated by a dashed line. Data are pooled from different experiments. Experiments were performed twice, n = 3–5 mice per experiment, except for panel F that included 3 experiments, n = 3–5 mice per experiment; ns, not significant. Statistical analyses for panel A were performed with Kruskal-Wallis test with Dunn’s multiple comparison test; for panels B-D and G-H ANOVA for multiple comparisons with Holm-Sidak’s correction was used; for panels F and I, Wilcoxon matched-pairs signed rank test was used comparing pre-treatment viremia versus day 30 viremia within the same treatment group. Error bars represent SEM.

Techniques Used: Expressing, FACS, Mouse Assay

IFN- I signaling is dispensable for the potentiation of PD-1 therapy by LPS. ( A ) Systemic IFNα levels after LPS administration in chronically infected mice. Chronically infected mice (~day 45 post-infection) were treated with either PBS or LPS, and IFNα levels were quantified in sera after 24 hours. ( B ) Experimental outline for blocking interferon type I receptor. Mice chronically infected with LCMV Cl-13 received a standard PD-L1 blockade regimen combined with LPS administration and injection of IFNAR1 (MAR1-5A3) blocking antibody. ( C ) Representative FACS histogram corroborating that MAR1-5A3 antibody blocks the IFNAR1 receptor at day 3 post-treatment (gated on PBMCs). ( D ) Summary of D b GP276+ responses in spleen. ( E ) Summary of viral control in spleen. ( F ) Summary of Treg responses in spleen. ( G ) Representative FACS plots showing the frequencies of splenic CD4 T cells that are FoxP3+ (first column). In the second and third columns, FoxP3+ CD4 T cells were gated to quantify inhibitory receptor expression. Note that LPS treatment does not attenuate Treg responses. For plaque assays the limit of detection is indicated by a dashed line. Data are pooled from different experiments. Experiments were performed 2 times, n = 4–5 mice per experiment; ns, not significant. Statistical analyses were performed using ANOVA for multiple comparisons with Holm-Sidak’s correction. Error bars represent SEM.
Figure Legend Snippet: IFN- I signaling is dispensable for the potentiation of PD-1 therapy by LPS. ( A ) Systemic IFNα levels after LPS administration in chronically infected mice. Chronically infected mice (~day 45 post-infection) were treated with either PBS or LPS, and IFNα levels were quantified in sera after 24 hours. ( B ) Experimental outline for blocking interferon type I receptor. Mice chronically infected with LCMV Cl-13 received a standard PD-L1 blockade regimen combined with LPS administration and injection of IFNAR1 (MAR1-5A3) blocking antibody. ( C ) Representative FACS histogram corroborating that MAR1-5A3 antibody blocks the IFNAR1 receptor at day 3 post-treatment (gated on PBMCs). ( D ) Summary of D b GP276+ responses in spleen. ( E ) Summary of viral control in spleen. ( F ) Summary of Treg responses in spleen. ( G ) Representative FACS plots showing the frequencies of splenic CD4 T cells that are FoxP3+ (first column). In the second and third columns, FoxP3+ CD4 T cells were gated to quantify inhibitory receptor expression. Note that LPS treatment does not attenuate Treg responses. For plaque assays the limit of detection is indicated by a dashed line. Data are pooled from different experiments. Experiments were performed 2 times, n = 4–5 mice per experiment; ns, not significant. Statistical analyses were performed using ANOVA for multiple comparisons with Holm-Sidak’s correction. Error bars represent SEM.

Techniques Used: Infection, Mouse Assay, Blocking Assay, Injection, FACS, Expressing

9) Product Images from "CD45RB Glycosylation and Ig Isotype Define Maturation of Functionally Distinct B Cell Subsets in Human Peripheral Blood"

Article Title: CD45RB Glycosylation and Ig Isotype Define Maturation of Functionally Distinct B Cell Subsets in Human Peripheral Blood

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2022.891316

CD45RB glycosylation marks antigen-primed B cells independent of CD27 expression. (A) Representative gating strategy for cell-sorting B cell subsets from healthy donor peripheral blood CD19+ pre-isolated B cells by flow cytometry. Gated from singlet viable CD19+ cells ( Figure S1B ). Table shows the classification of subsets. (B) Frequency of subsets within CD19+ cells, for seven biological replicates each composed of two technical replicates over three independent experiments. (C) Mean number of mutations per Ig gene (healthy adults, n = 3) and (D) Cumulative frequency distribution of mutations in the Ig repertoire in the different B cell subsets. Different colored lines represent different donors. (E) mean V H -CDR3 length from cell-sorted B cell subsets (healthy adults, n = 3). Bars and black lines depict mean values. Statistical differences were determined using a Friedman analysis of variance and Dunn’s multiple comparison test. *p
Figure Legend Snippet: CD45RB glycosylation marks antigen-primed B cells independent of CD27 expression. (A) Representative gating strategy for cell-sorting B cell subsets from healthy donor peripheral blood CD19+ pre-isolated B cells by flow cytometry. Gated from singlet viable CD19+ cells ( Figure S1B ). Table shows the classification of subsets. (B) Frequency of subsets within CD19+ cells, for seven biological replicates each composed of two technical replicates over three independent experiments. (C) Mean number of mutations per Ig gene (healthy adults, n = 3) and (D) Cumulative frequency distribution of mutations in the Ig repertoire in the different B cell subsets. Different colored lines represent different donors. (E) mean V H -CDR3 length from cell-sorted B cell subsets (healthy adults, n = 3). Bars and black lines depict mean values. Statistical differences were determined using a Friedman analysis of variance and Dunn’s multiple comparison test. *p

Techniques Used: Expressing, FACS, Isolation, Flow Cytometry

CD45RB glycosylation, combined with Ig isotype, marks functionally distinct B cell subsets. B cell subsets were cultured with TD-stimulation of CD40L-expressing 3T3s in the presence of IL-21 (50ng/ml) for 10 days (six biological replicates each consisting of two technical replicates over two independent experiments). (A) Representative biaxial CD27/CD38 FACS plot after 10 days of TD culture. The formation of (B) MBCs (CD27+CD38-) (C) IgG+ B cells, and (D) ASCs (CD27+CD38+) was measured using flow cytometry. (E) Cumulative IgM secretion measured in culture supernatants using ELISA at day 10 (n = 6). B cell subsets were cultured with TI-stimulation of CpG ODN (0.1μM) and anti-IgM-F(ab’) 2 (1μg/ml) for 7 days (four biological replicates each consisting of two technical replicates). The formation of (F) MBCs and (G) ASCs was measured using flow cytometry. Black lines depict mean values. Statistical differences were determined using a Friedman analysis of variance and Dunn’s multiple comparison test. *p
Figure Legend Snippet: CD45RB glycosylation, combined with Ig isotype, marks functionally distinct B cell subsets. B cell subsets were cultured with TD-stimulation of CD40L-expressing 3T3s in the presence of IL-21 (50ng/ml) for 10 days (six biological replicates each consisting of two technical replicates over two independent experiments). (A) Representative biaxial CD27/CD38 FACS plot after 10 days of TD culture. The formation of (B) MBCs (CD27+CD38-) (C) IgG+ B cells, and (D) ASCs (CD27+CD38+) was measured using flow cytometry. (E) Cumulative IgM secretion measured in culture supernatants using ELISA at day 10 (n = 6). B cell subsets were cultured with TI-stimulation of CpG ODN (0.1μM) and anti-IgM-F(ab’) 2 (1μg/ml) for 7 days (four biological replicates each consisting of two technical replicates). The formation of (F) MBCs and (G) ASCs was measured using flow cytometry. Black lines depict mean values. Statistical differences were determined using a Friedman analysis of variance and Dunn’s multiple comparison test. *p

Techniques Used: Cell Culture, Expressing, FACS, Flow Cytometry, Enzyme-linked Immunosorbent Assay

10) Product Images from "Systematic functional screening of chromatin factors identifies strong lineage and disease dependencies in normal and malignant haematopoiesis"

Article Title: Systematic functional screening of chromatin factors identifies strong lineage and disease dependencies in normal and malignant haematopoiesis

Journal: bioRxiv

doi: 10.1101/2022.08.11.503571

Functional interrogation of Chromatin Factors (CF) reveals strong lineage dependencies during haematopoietic differentiation. ( a ) Schematic drawing of the CRISPR screen workflow (left to right)- Isolation of Multipotent progenitors (Lin-, Sca1+, ckit+) from Cas9-GFP and Non-Cas9 mouse strains; Delivery of the CRISPR library targeting 550 Chromatin Factors to the mixed Cas9 and non-Cas9 population of multipotent progenitors ; Differentiation of CRISPR modified multipotent progenitors using 4 ex vivo culture systems (details in panel 1b); FACS-based differentiation readouts (see panel 1b) from Cas9 and Non-Cas9 fractions; Quantification of sgRNA distributions across the readout populations ; Calculation of lineage scores for each Chromatin Factor, using the Non-Cas9 sgRNA distributions as a background model to correct for library bias and dispersion. (b) Differentiation systems and FACS-based Readouts. 1- Self-Renewal vs Differentiation : culture driven by SCF and Tpo where Multipotent (Lin-, ckit+, Sca1+) and Differentiated (Lin-, ckit+, Sca1-) populations are evaluated. 2- Lineage priming : culture driven by SCF, Flt3L, and Tpo where Mega-erythroid (Lin-, ckit+, Sca1-, FcgR- III-) and Myeloid (Lin-, ckit+, Sca1-, FcgR-III+) progenitors are evaluated. 3 - Myeloid Differentiation : culture driven by GM-CSF, G-CSF, Flt3L, IL3 and EPO where Mature myeloid (FcgR-III+, CD11b+) and Non-Myeloid (FcgR-III-, CD11b-) lineages are evaluated. 4 - Terminal Myeloid Maturation : culture driven by GM-CSF, G-CSF, Flt3L, IL3 where Immature myeloid (CD11b-, Gr1-) and Mature myeloid (CD11b+, Gr1+) lineages are evaluated. Screens 1-3 are initiated from LSK and screen 4 from committed myeloid progenitors (Granulocyte macrophage progenitors, GMPs). (c) 2D Projection of lineage scores for “1- Differentiation vs Self-renewal” (y-axis) and “2- Lineage priming” into myeloid vs mega-erythroid (x-axis). For each gene, lineage scores are shown aggregated across all guides and libraries. Genes with significant changes are shown with a red cross. Genes that did not reach significance thresholds are shown as black dots, aggregated NTCs are shown with a blue cross. Data for Non-Cas9 cells are shown in the background using a yellow-blue density. ( d ) Lineage scores for chromatin factors grouped on the basis of complex membership. The colour of each dot represents the lineage score. The size of the dot represents the number of significant guides. ( e ) Exemplar immunophenotypic validations for CFs with strong scores in the lineage priming (top) and myeloid differentiation (bottom) systems.
Figure Legend Snippet: Functional interrogation of Chromatin Factors (CF) reveals strong lineage dependencies during haematopoietic differentiation. ( a ) Schematic drawing of the CRISPR screen workflow (left to right)- Isolation of Multipotent progenitors (Lin-, Sca1+, ckit+) from Cas9-GFP and Non-Cas9 mouse strains; Delivery of the CRISPR library targeting 550 Chromatin Factors to the mixed Cas9 and non-Cas9 population of multipotent progenitors ; Differentiation of CRISPR modified multipotent progenitors using 4 ex vivo culture systems (details in panel 1b); FACS-based differentiation readouts (see panel 1b) from Cas9 and Non-Cas9 fractions; Quantification of sgRNA distributions across the readout populations ; Calculation of lineage scores for each Chromatin Factor, using the Non-Cas9 sgRNA distributions as a background model to correct for library bias and dispersion. (b) Differentiation systems and FACS-based Readouts. 1- Self-Renewal vs Differentiation : culture driven by SCF and Tpo where Multipotent (Lin-, ckit+, Sca1+) and Differentiated (Lin-, ckit+, Sca1-) populations are evaluated. 2- Lineage priming : culture driven by SCF, Flt3L, and Tpo where Mega-erythroid (Lin-, ckit+, Sca1-, FcgR- III-) and Myeloid (Lin-, ckit+, Sca1-, FcgR-III+) progenitors are evaluated. 3 - Myeloid Differentiation : culture driven by GM-CSF, G-CSF, Flt3L, IL3 and EPO where Mature myeloid (FcgR-III+, CD11b+) and Non-Myeloid (FcgR-III-, CD11b-) lineages are evaluated. 4 - Terminal Myeloid Maturation : culture driven by GM-CSF, G-CSF, Flt3L, IL3 where Immature myeloid (CD11b-, Gr1-) and Mature myeloid (CD11b+, Gr1+) lineages are evaluated. Screens 1-3 are initiated from LSK and screen 4 from committed myeloid progenitors (Granulocyte macrophage progenitors, GMPs). (c) 2D Projection of lineage scores for “1- Differentiation vs Self-renewal” (y-axis) and “2- Lineage priming” into myeloid vs mega-erythroid (x-axis). For each gene, lineage scores are shown aggregated across all guides and libraries. Genes with significant changes are shown with a red cross. Genes that did not reach significance thresholds are shown as black dots, aggregated NTCs are shown with a blue cross. Data for Non-Cas9 cells are shown in the background using a yellow-blue density. ( d ) Lineage scores for chromatin factors grouped on the basis of complex membership. The colour of each dot represents the lineage score. The size of the dot represents the number of significant guides. ( e ) Exemplar immunophenotypic validations for CFs with strong scores in the lineage priming (top) and myeloid differentiation (bottom) systems.

Techniques Used: Functional Assay, CRISPR, Isolation, Modification, Ex Vivo, FACS

11) Product Images from "Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy"

Article Title: Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy

Journal: Nature Communications

doi: 10.1038/s41467-021-21168-6

CD19 neg B-ALL relapse following CAR-T therapy. a Cell sorting strategy. Cells before (T1) and after (T2) CAR-T treatment were gated according to FSC/SSC profile (left FACS plots). Then, live cells (middle FACS plots) were analyzed according to CD3 and CD19 expression (right FACS plots; see also Supplementary Fig. 9 ). Gates of the four sorted subpopulations T1-CD19 pos , T1-CD19 neg , T2-CD19 pos , and T2-CD19 neg are highlighted in cyan, red, green, and gold, respectively. Those sorted subpopulations were labeled with a specific anti-CD45-HTO antibody, then multiplexed and analyzed by scRNAseq using the 10× Genomics single-cell 5′ technology. Some cells from T1-CD19 pos and T2-CD19 neg samples were used in bulk to prepare cDNA. b Seurat Dotplot showing the expression level of marker genes in each cluster. Dot size represents the percentage of cell expressing the gene of interest, while dot color represents the scaled average expression (Scaled Avg. Exp.) of the gene of interest across the various clusters (a negative value corresponds to an expression below the mean expression). We used CD34 and RPS14 expression as tumoral markers. Indeed FISH analysis revealed a 5q32 deletion, explaining RPS14 lower expression in B-ALL cells. c UMAP visualization of the six main clusters and their corresponding cell types of T1 and T2 sorted samples. d UMAP visualization of the four demultiplexed samples: T1-CD19 neg , T1-CD19 pos , T2-CD19 neg , and T2-CD19 pos . e Agarose gel of CD19 cDNA amplified products using exons-specific primer sets depicted on the top panel. PCR were performed with bulk cDNA from T1-CD19 pos cells and T2-CD19 neg cells. Agarose gel data are representative of two independent experiments. Lane “Lad” is the 1 kb DNA size marker. Schematic representations of PCR products indicated by “a”–“f” arrows are shown below the gel.
Figure Legend Snippet: CD19 neg B-ALL relapse following CAR-T therapy. a Cell sorting strategy. Cells before (T1) and after (T2) CAR-T treatment were gated according to FSC/SSC profile (left FACS plots). Then, live cells (middle FACS plots) were analyzed according to CD3 and CD19 expression (right FACS plots; see also Supplementary Fig. 9 ). Gates of the four sorted subpopulations T1-CD19 pos , T1-CD19 neg , T2-CD19 pos , and T2-CD19 neg are highlighted in cyan, red, green, and gold, respectively. Those sorted subpopulations were labeled with a specific anti-CD45-HTO antibody, then multiplexed and analyzed by scRNAseq using the 10× Genomics single-cell 5′ technology. Some cells from T1-CD19 pos and T2-CD19 neg samples were used in bulk to prepare cDNA. b Seurat Dotplot showing the expression level of marker genes in each cluster. Dot size represents the percentage of cell expressing the gene of interest, while dot color represents the scaled average expression (Scaled Avg. Exp.) of the gene of interest across the various clusters (a negative value corresponds to an expression below the mean expression). We used CD34 and RPS14 expression as tumoral markers. Indeed FISH analysis revealed a 5q32 deletion, explaining RPS14 lower expression in B-ALL cells. c UMAP visualization of the six main clusters and their corresponding cell types of T1 and T2 sorted samples. d UMAP visualization of the four demultiplexed samples: T1-CD19 neg , T1-CD19 pos , T2-CD19 neg , and T2-CD19 pos . e Agarose gel of CD19 cDNA amplified products using exons-specific primer sets depicted on the top panel. PCR were performed with bulk cDNA from T1-CD19 pos cells and T2-CD19 neg cells. Agarose gel data are representative of two independent experiments. Lane “Lad” is the 1 kb DNA size marker. Schematic representations of PCR products indicated by “a”–“f” arrows are shown below the gel.

Techniques Used: FACS, Expressing, Labeling, Marker, Fluorescence In Situ Hybridization, Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction

12) Product Images from "RORγt+ cells selectively express redundant cation channels linked to the Golgi apparatus"

Article Title: RORγt+ cells selectively express redundant cation channels linked to the Golgi apparatus

Journal: Scientific Reports

doi: 10.1038/srep23682

Tmem176b single-deficient mouse susceptibility to the development of EAE, chronic and atute colitis. ( a ) EAE was induced in WT (n = 6) and Tmem176b −/− (n = 8) mice by immunisation (s.c.) with MOG peptide in CFA. Clinical course of disease is shown. ( b ) Chronic colitis was induced in Rag1 −/− mice (n = 11–12 in each group) by adoptive transfer (i.v.) of FACS-sorted CD4 + CD45RB hi T cells from WT or Tmem176b −/− mice. Data are presented as percent of initial weight. ( c ) Acute colitis was induced in WT (n = 6) and Tmem176b −/− (n = 8) mice with 3% DSS in drinking water for 5 consecutive days. Data are presented as percent of initial weight.
Figure Legend Snippet: Tmem176b single-deficient mouse susceptibility to the development of EAE, chronic and atute colitis. ( a ) EAE was induced in WT (n = 6) and Tmem176b −/− (n = 8) mice by immunisation (s.c.) with MOG peptide in CFA. Clinical course of disease is shown. ( b ) Chronic colitis was induced in Rag1 −/− mice (n = 11–12 in each group) by adoptive transfer (i.v.) of FACS-sorted CD4 + CD45RB hi T cells from WT or Tmem176b −/− mice. Data are presented as percent of initial weight. ( c ) Acute colitis was induced in WT (n = 6) and Tmem176b −/− (n = 8) mice with 3% DSS in drinking water for 5 consecutive days. Data are presented as percent of initial weight.

Techniques Used: Mouse Assay, Adoptive Transfer Assay, FACS

Tmem176b single-deficient mouse susceptibility to the development of psoriasis-like dermatitis. ( a ) Psoriasis-like dermatitis was induced in WT mice by topical application of imiquimod (IMQ) cream on the shaved back skin. At day 4, draining (inguinal) lymph nodes were harvested and TCRγδ + and TCRβ + CD4 + T cells were FACS-sorted. ( b ) Expression of indicated genes was assessed by quantitative RT-PCR. Each dot represents an individual mouse (n = 6 in each group). Statistically significant differences are indicated: **p
Figure Legend Snippet: Tmem176b single-deficient mouse susceptibility to the development of psoriasis-like dermatitis. ( a ) Psoriasis-like dermatitis was induced in WT mice by topical application of imiquimod (IMQ) cream on the shaved back skin. At day 4, draining (inguinal) lymph nodes were harvested and TCRγδ + and TCRβ + CD4 + T cells were FACS-sorted. ( b ) Expression of indicated genes was assessed by quantitative RT-PCR. Each dot represents an individual mouse (n = 6 in each group). Statistically significant differences are indicated: **p

Techniques Used: Mouse Assay, FACS, Expressing, Quantitative RT-PCR

Tmem176a and b mRNA expression in mouse and human T cells. ( a ) Conventional GFP − (Foxp3 − Tconv) or regulatory GFP + (Foxp3 + Treg) CD4 + T cells were FACS-sorted from the spleen or intestinal lamina propria (small intestine and colon) of Foxp3 EGFP mice. As expected, the population of Nrp1 −/low “adaptive” peripherally Tregs is dominant in the intestines. Conversely, Nrp1 + “natural” thymically derived Tregs represent the major population of Tregs in spleen. ( b ) Expression of indicated genes was assessed by quantitative RT-PCR. Each dot represents an individual mouse (n = 6–7 in each group). Statistically significant differences between intestinal Tconv and Treg are indicated: *p
Figure Legend Snippet: Tmem176a and b mRNA expression in mouse and human T cells. ( a ) Conventional GFP − (Foxp3 − Tconv) or regulatory GFP + (Foxp3 + Treg) CD4 + T cells were FACS-sorted from the spleen or intestinal lamina propria (small intestine and colon) of Foxp3 EGFP mice. As expected, the population of Nrp1 −/low “adaptive” peripherally Tregs is dominant in the intestines. Conversely, Nrp1 + “natural” thymically derived Tregs represent the major population of Tregs in spleen. ( b ) Expression of indicated genes was assessed by quantitative RT-PCR. Each dot represents an individual mouse (n = 6–7 in each group). Statistically significant differences between intestinal Tconv and Treg are indicated: *p

Techniques Used: Expressing, FACS, Mouse Assay, Derivative Assay, Quantitative RT-PCR

13) Product Images from "Allergen recognition by specific effector Th2 cells enables IL-2-dependent activation of regulatory T cell responses in humans"

Article Title: Allergen recognition by specific effector Th2 cells enables IL-2-dependent activation of regulatory T cell responses in humans

Journal: medRxiv

doi: 10.1101/2022.05.17.22275017

T cell responses to peanut allergens are associated with the activation of unique subsets of highly differentiated effector Th2 cells and memory regulatory T cells (Tregs) in peanut allergic (PA) subjects. A . Overlap of the activated populations after stimulation with crude peanut extract (CPE) for 6h, 24h, and 48h represented by Venn diagrams (n=24-30). Numbers inside the circles indicate the percentage (± SEM) of non-overlapped cells and numbers next to the lines specify the overlap percentage (± SEM). Different colors designate different activated populations. B . Estimated Chao1 alpha diversity of TCRβ repertoire in activated populations and total memory CD4 + T cells sorted from PA subjects stimulated with CPE for 6h and 24h (n=3). C . Percentage of the mature effector ( m T EM ), effector (T EM ), and central (T CM ) memory CD4 + T cells in the activated populations after stimulation of PBMCs from PA subjects with CPE for 6h, 24h, and 48h (n=25). D . Percentage of chemokine receptor co-expression in activated populations after CPE-stimulation for 6h, 24h, and 48h (n=5). E . Heatmaps of the percentage of expression for each marker in Foxp3 - T cells (left) and Tregs (Foxp3 + , right) in activated populations after CPE-stimulation for 6h, 24h, and 48h (n=5). In (B) and (C) each data point is one individual (mean ± SEM) and bar graphs represent mean + SEM in (D). Statistical analyses by mixed-effect analysis with Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test in (B) and (C). In (D), dashed line represents cut off above populations are significantly increased (mixed-effect analysis accounting for subject levels repeated measures at the three timepoints studied with Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test, p
Figure Legend Snippet: T cell responses to peanut allergens are associated with the activation of unique subsets of highly differentiated effector Th2 cells and memory regulatory T cells (Tregs) in peanut allergic (PA) subjects. A . Overlap of the activated populations after stimulation with crude peanut extract (CPE) for 6h, 24h, and 48h represented by Venn diagrams (n=24-30). Numbers inside the circles indicate the percentage (± SEM) of non-overlapped cells and numbers next to the lines specify the overlap percentage (± SEM). Different colors designate different activated populations. B . Estimated Chao1 alpha diversity of TCRβ repertoire in activated populations and total memory CD4 + T cells sorted from PA subjects stimulated with CPE for 6h and 24h (n=3). C . Percentage of the mature effector ( m T EM ), effector (T EM ), and central (T CM ) memory CD4 + T cells in the activated populations after stimulation of PBMCs from PA subjects with CPE for 6h, 24h, and 48h (n=25). D . Percentage of chemokine receptor co-expression in activated populations after CPE-stimulation for 6h, 24h, and 48h (n=5). E . Heatmaps of the percentage of expression for each marker in Foxp3 - T cells (left) and Tregs (Foxp3 + , right) in activated populations after CPE-stimulation for 6h, 24h, and 48h (n=5). In (B) and (C) each data point is one individual (mean ± SEM) and bar graphs represent mean + SEM in (D). Statistical analyses by mixed-effect analysis with Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test in (B) and (C). In (D), dashed line represents cut off above populations are significantly increased (mixed-effect analysis accounting for subject levels repeated measures at the three timepoints studied with Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test, p

Techniques Used: Activation Assay, Expressing, Marker

Peanut allergens induce an early T cell response driven by CD154 + CD69 + cells and a delayed activation of suppressive regulatory T cells (Tregs) in peanut allergic (PA) subjects. A . Minimal spanning tree visualization of FlowSOM clustering analysis of memory (CD45RA) - CD4 + T cells of PA subjects stimulated with crude peanut extract (CPE) for 6h, 24h, and 48h (n=5). Different nodes indicate the relative size of the cluster identified. Meta-clusters (MC) are indicated with different numbers. B . Intensity of expression of activation markers visualized by a star chart in each node of the FlowSOM clustering analysis (n=5). Each pie height indicates intensity of expression. C . Percentage of the activated populations identified in memory CD4 + T cells unstimulated (CTRL) or stimulated with CPE for 6h, 24h, and 48h (n=30). D . Percentage of Foxp3 - (white) and Foxp3 + (green) CD4 + T cells in the activated populations after CPE-stimulation for 6h, 24h, and 48h (n=13-14). E . Inhibition percentage of CD25 - CD4 + T cell proliferation induced by activated Tregs populations sorted from polyclonally activated PBMCs. Suppression of CD25 - CD4 + T cell proliferation was calculated via CFSE dilution. F . Quantification of IL-4 and IL-5 released by polarized Th2 cells alone or cultured with activated Treg populations sorted from polyclonally activated PBMCs for 72h. In (C) each data point is one individual (mean ± SEM), each point represents the mean ± SEM of three independent experiments performed in triplicate in (E), and bar graphs represent mean + SEM of three independent experiments performed in triplicate in (F). Statistical analyses by mixed-effect analysis with Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test in (C) and (E), and ordinary one-way ANOVA with Geisser-Greenhouse correction followed by Dunnett’s multiple comparisons test in (F). *P
Figure Legend Snippet: Peanut allergens induce an early T cell response driven by CD154 + CD69 + cells and a delayed activation of suppressive regulatory T cells (Tregs) in peanut allergic (PA) subjects. A . Minimal spanning tree visualization of FlowSOM clustering analysis of memory (CD45RA) - CD4 + T cells of PA subjects stimulated with crude peanut extract (CPE) for 6h, 24h, and 48h (n=5). Different nodes indicate the relative size of the cluster identified. Meta-clusters (MC) are indicated with different numbers. B . Intensity of expression of activation markers visualized by a star chart in each node of the FlowSOM clustering analysis (n=5). Each pie height indicates intensity of expression. C . Percentage of the activated populations identified in memory CD4 + T cells unstimulated (CTRL) or stimulated with CPE for 6h, 24h, and 48h (n=30). D . Percentage of Foxp3 - (white) and Foxp3 + (green) CD4 + T cells in the activated populations after CPE-stimulation for 6h, 24h, and 48h (n=13-14). E . Inhibition percentage of CD25 - CD4 + T cell proliferation induced by activated Tregs populations sorted from polyclonally activated PBMCs. Suppression of CD25 - CD4 + T cell proliferation was calculated via CFSE dilution. F . Quantification of IL-4 and IL-5 released by polarized Th2 cells alone or cultured with activated Treg populations sorted from polyclonally activated PBMCs for 72h. In (C) each data point is one individual (mean ± SEM), each point represents the mean ± SEM of three independent experiments performed in triplicate in (E), and bar graphs represent mean + SEM of three independent experiments performed in triplicate in (F). Statistical analyses by mixed-effect analysis with Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test in (C) and (E), and ordinary one-way ANOVA with Geisser-Greenhouse correction followed by Dunnett’s multiple comparisons test in (F). *P

Techniques Used: Activation Assay, Expressing, Inhibition, Cell Culture

14) Product Images from "SARS coronavirus vaccines protect against different coronaviruses"

Article Title: SARS coronavirus vaccines protect against different coronaviruses

Journal: bioRxiv

doi: 10.1101/2021.06.01.446491

Mapping of conserved CD8 T cell epitopes following SARS-CoV-2 vaccination. ( A ) Epitope mapping using Ad5-SARS-2 spike immune splenocytes (week 2 post-boost), stimulated with overlapping SARS-CoV-2 spike peptide pools for 5 hr at 37°C in a CO 2 incubator. This study identified 2 potential K b binding epitopes that are highly conserved among multiple coronaviruses, a subdominant VVLSFELL epitope and a dominant VNFNFNGL epitope. ( B ) K b VNFNFNGL (K b VL8) tetramer+ CD8 T cells were FACS-sorted for TCR-sequencing (week 4 post-prime). ( C ) Top 5 TCR usages (percent of total VL8-specific). ( D ) TCR sequences in VL8-specific CD8 T cells. Data from panel A represent a pooled sample from 5 spleens. Data from panels B-D are from 1 experiment with 1 mouse.
Figure Legend Snippet: Mapping of conserved CD8 T cell epitopes following SARS-CoV-2 vaccination. ( A ) Epitope mapping using Ad5-SARS-2 spike immune splenocytes (week 2 post-boost), stimulated with overlapping SARS-CoV-2 spike peptide pools for 5 hr at 37°C in a CO 2 incubator. This study identified 2 potential K b binding epitopes that are highly conserved among multiple coronaviruses, a subdominant VVLSFELL epitope and a dominant VNFNFNGL epitope. ( B ) K b VNFNFNGL (K b VL8) tetramer+ CD8 T cells were FACS-sorted for TCR-sequencing (week 4 post-prime). ( C ) Top 5 TCR usages (percent of total VL8-specific). ( D ) TCR sequences in VL8-specific CD8 T cells. Data from panel A represent a pooled sample from 5 spleens. Data from panels B-D are from 1 experiment with 1 mouse.

Techniques Used: Binding Assay, FACS, Sequencing

SARS-CoV-1 vaccination induces cross-reactive antibodies and T cells. ( A ) Antibody responses after MVA-SARS-CoV-1 spike vaccination. ( B ) SARS-CoV-2 pseudovirus neutralization assay. ( C ) Representative microscopy image of SARS-CoV-2 pseudovirus neutralization using sera from unvaccinated mice. ( D ) Representative microscopy image of SARS-CoV-2 pseudovirus neutralization using sera from SARS-CoV-1 vaccinated mice. ( E ) Representative FACS plots showing cross-reactive SARS-CoV-2 specific CD8 T cells in SARS- CoV-1 vaccinated mice. Cross-reactive CD8 T cells were detected by intracellular cytokine staining after 5 hr stimulation with SARS-CoV-2 spike overlapping peptide pools, in a 37°C 5% CO 2 incubator. Cells are gated from total live CD8 T cells in spleen (week 2 post-boost). ( F ) Representative FACS plots showing cross-reactive (VNFNFNGL-specific) CD8 T cells in mice vaccinated with a SARS-CoV-1 vaccine, and various SARS-CoV-2 vaccines (Ad5-based, VSV-based and mRNA-based). Note that the K b VNFNFNGL (K b VL8) tetramer could be used to identify cross-reactive CD8 T cell responses among multiple vaccine platforms. Cells are gated from total live CD8 T cells in PBMCs (day 15 post-boost). ( G ) Summary of cross-reactive (VNFNFNGL- specific) CD8 T cells in mice vaccinated with a SARS-CoV-1 vaccine, and various SARS-CoV-2 vaccines. All mice were primed and boosted intramuscularly (see Materials and Methods for vaccine dosing information). Arrows in panel G indicate time of boosting. Experiments were done using wild type C57BL/6 mice, except for VSV-SARS-2 spike vaccination, which used k18- hACE2 (C57BL/6) mice. Dashed lines represent limit of detection. Data are from 1 representative experiment with n=5/group; experiments were performed 2-3 times with similar results. **, P
Figure Legend Snippet: SARS-CoV-1 vaccination induces cross-reactive antibodies and T cells. ( A ) Antibody responses after MVA-SARS-CoV-1 spike vaccination. ( B ) SARS-CoV-2 pseudovirus neutralization assay. ( C ) Representative microscopy image of SARS-CoV-2 pseudovirus neutralization using sera from unvaccinated mice. ( D ) Representative microscopy image of SARS-CoV-2 pseudovirus neutralization using sera from SARS-CoV-1 vaccinated mice. ( E ) Representative FACS plots showing cross-reactive SARS-CoV-2 specific CD8 T cells in SARS- CoV-1 vaccinated mice. Cross-reactive CD8 T cells were detected by intracellular cytokine staining after 5 hr stimulation with SARS-CoV-2 spike overlapping peptide pools, in a 37°C 5% CO 2 incubator. Cells are gated from total live CD8 T cells in spleen (week 2 post-boost). ( F ) Representative FACS plots showing cross-reactive (VNFNFNGL-specific) CD8 T cells in mice vaccinated with a SARS-CoV-1 vaccine, and various SARS-CoV-2 vaccines (Ad5-based, VSV-based and mRNA-based). Note that the K b VNFNFNGL (K b VL8) tetramer could be used to identify cross-reactive CD8 T cell responses among multiple vaccine platforms. Cells are gated from total live CD8 T cells in PBMCs (day 15 post-boost). ( G ) Summary of cross-reactive (VNFNFNGL- specific) CD8 T cells in mice vaccinated with a SARS-CoV-1 vaccine, and various SARS-CoV-2 vaccines. All mice were primed and boosted intramuscularly (see Materials and Methods for vaccine dosing information). Arrows in panel G indicate time of boosting. Experiments were done using wild type C57BL/6 mice, except for VSV-SARS-2 spike vaccination, which used k18- hACE2 (C57BL/6) mice. Dashed lines represent limit of detection. Data are from 1 representative experiment with n=5/group; experiments were performed 2-3 times with similar results. **, P

Techniques Used: Neutralization, Microscopy, Mouse Assay, FACS, Staining

A T cell-based SARS vaccine protects against a distant coronavirus challenge. ( A ) Experiment outline. C57BL/6 mice received DC2.4 cells coated with SARS-CoV-2 peptide pools derived from s pike, e nvelope, m embrane, and n ucleocapsid proteins (DC-SEMN). After a week, mice were boosted with splenocytes coated with these same peptide pools. DMSO vehicle-coated cells were used as controls (see Materials and Methods). A week after boost, mice were challenged intranasally with MHV-1, and viral loads were assessed in lungs at day 3 by plaque assays. ( B ) Representative FACS plots showing SARS-CoV-2 SEMN-specific CD8 T cells. SARS-CoV-2 specific T cells were detected by intracellular cytokine staining after 5 hr stimulation with SARS- CoV-2 overlapping peptide pools (SEMN), in a 37°C 5% CO 2 incubator. Cells are gated from total live CD8 T cells in spleen (week 1 post-boost). ( C ) Summary of SARS-CoV-2 specific CD8 T cell responses. ( D ) SARS-CoV-2 spike-specific antibody. ( E ) Viral loads after MHV-1 challenge. SARS-CoV-2 and MHV-1 share ∼38% identity in SEMN. Dashed lines represent limit of detection. Experiment was performed two times, with n=4-5 mice per group, per experiment. Data from all experiments are shown. P value is indicated (Mann Whitney U Test). **, P
Figure Legend Snippet: A T cell-based SARS vaccine protects against a distant coronavirus challenge. ( A ) Experiment outline. C57BL/6 mice received DC2.4 cells coated with SARS-CoV-2 peptide pools derived from s pike, e nvelope, m embrane, and n ucleocapsid proteins (DC-SEMN). After a week, mice were boosted with splenocytes coated with these same peptide pools. DMSO vehicle-coated cells were used as controls (see Materials and Methods). A week after boost, mice were challenged intranasally with MHV-1, and viral loads were assessed in lungs at day 3 by plaque assays. ( B ) Representative FACS plots showing SARS-CoV-2 SEMN-specific CD8 T cells. SARS-CoV-2 specific T cells were detected by intracellular cytokine staining after 5 hr stimulation with SARS- CoV-2 overlapping peptide pools (SEMN), in a 37°C 5% CO 2 incubator. Cells are gated from total live CD8 T cells in spleen (week 1 post-boost). ( C ) Summary of SARS-CoV-2 specific CD8 T cell responses. ( D ) SARS-CoV-2 spike-specific antibody. ( E ) Viral loads after MHV-1 challenge. SARS-CoV-2 and MHV-1 share ∼38% identity in SEMN. Dashed lines represent limit of detection. Experiment was performed two times, with n=4-5 mice per group, per experiment. Data from all experiments are shown. P value is indicated (Mann Whitney U Test). **, P

Techniques Used: Mouse Assay, Derivative Assay, FACS, Staining, MANN-WHITNEY

15) Product Images from "P2RY14 cAMP signaling regulates Schwann cell precursor self-renewal, proliferation, and nerve tumor initiation in a mouse model of neurofibromatosis"

Article Title: P2RY14 cAMP signaling regulates Schwann cell precursor self-renewal, proliferation, and nerve tumor initiation in a mouse model of neurofibromatosis

Journal: eLife

doi: 10.7554/eLife.73511

P2RY14 is expressed in human neurofibromas and promotes Schwann cell precursor (SCP) self-renewal in vitro. ( A ) Microarray heatmap shows P2ry14 receptor expression in p75 + /EGFR + SCP-like tumor-initiating cells derived from human plexiform neurofibroma tumor cells compared to p75 + /EGFR - SCP-like cells. ( B ) Western blot of human Schwann cells and neurofibroma SCP shows the latter has a 1.9-fold increase in P2ry14 protein expression. ( C ) Immunohistochemistry of human neurofibroma shows P2ry14 expression (DAB staining: brown [P2ry14 positive cells] blue [cell nuclei]). ( D ) Representative fluorescence-activated cells sorting (FACS) plot shows live sorted human plexiform neurofibroma tumor cells. ( E ) Representative FACS plot shows human plexiform neurofibroma tumor cells sorted into p75 + /EGFR + SCP-like tumor-initiating cells (pink square). ( F ) Representative FACS plot shows p75 + /EGFR + SCP-like tumor-initiating cells further sorted into p75 + /EGFR + / P2ry14 - (left, purple square) and P75 + /EGFR + / P2ry14 + (right, blue square). ( G ) Photomicrographs of human neurofibromas dissociated using FACS to yield: unsorted, p75 + /EGFR + / P2ry14 - and P75 + /EGFR + / P2ry14 + cells. ( H ) Quantification of unsorted, p75 + /EGFR + / P2ry14 - and P75 + /EGFR + / P2ry14 + cells plated in sphere medium. (n = 3; two-way ANOVA; primary: **p = 0.0057, ****p
Figure Legend Snippet: P2RY14 is expressed in human neurofibromas and promotes Schwann cell precursor (SCP) self-renewal in vitro. ( A ) Microarray heatmap shows P2ry14 receptor expression in p75 + /EGFR + SCP-like tumor-initiating cells derived from human plexiform neurofibroma tumor cells compared to p75 + /EGFR - SCP-like cells. ( B ) Western blot of human Schwann cells and neurofibroma SCP shows the latter has a 1.9-fold increase in P2ry14 protein expression. ( C ) Immunohistochemistry of human neurofibroma shows P2ry14 expression (DAB staining: brown [P2ry14 positive cells] blue [cell nuclei]). ( D ) Representative fluorescence-activated cells sorting (FACS) plot shows live sorted human plexiform neurofibroma tumor cells. ( E ) Representative FACS plot shows human plexiform neurofibroma tumor cells sorted into p75 + /EGFR + SCP-like tumor-initiating cells (pink square). ( F ) Representative FACS plot shows p75 + /EGFR + SCP-like tumor-initiating cells further sorted into p75 + /EGFR + / P2ry14 - (left, purple square) and P75 + /EGFR + / P2ry14 + (right, blue square). ( G ) Photomicrographs of human neurofibromas dissociated using FACS to yield: unsorted, p75 + /EGFR + / P2ry14 - and P75 + /EGFR + / P2ry14 + cells. ( H ) Quantification of unsorted, p75 + /EGFR + / P2ry14 - and P75 + /EGFR + / P2ry14 + cells plated in sphere medium. (n = 3; two-way ANOVA; primary: **p = 0.0057, ****p

Techniques Used: In Vitro, Microarray, Expressing, Derivative Assay, Western Blot, Immunohistochemistry, Staining, Fluorescence, FACS

16) Product Images from "Tissue-specific emergence of regulatory and intraepithelial T cells from a clonal T cell precursor"

Article Title: Tissue-specific emergence of regulatory and intraepithelial T cells from a clonal T cell precursor

Journal: Science immunology

doi: 10.1126/sciimmunol.aaf7471

Gene expression analysis of pT reg and CD4 IEL TN cells. TCRαβKO mice were transferred with 5 × 10 5 naïve CD4 + CD8α - Foxp3-GFP - T cells purified from the spleen of pT reg TN/RKO/Foxp3 gfp mice. Small intestine lymphocytes were harvested 6 weeks after transfer, and the indicated donor T cell subsets were sorted for RNA-seq library preparation and sequencing. ( A ) Gating strategy for FACS sorting of the indicated populations. ( B ) Principal components analysis of the top 300 differentially expressed genes by plotting contribution of each T cell population to the first two principal components (PC1 and PC2) (left). Gene set enrichment analysis (GSEA) of Foxp3 + pT regs versus “rest” within each intestinal compartment defined by the PC2 shows significant enrichment ( P
Figure Legend Snippet: Gene expression analysis of pT reg and CD4 IEL TN cells. TCRαβKO mice were transferred with 5 × 10 5 naïve CD4 + CD8α - Foxp3-GFP - T cells purified from the spleen of pT reg TN/RKO/Foxp3 gfp mice. Small intestine lymphocytes were harvested 6 weeks after transfer, and the indicated donor T cell subsets were sorted for RNA-seq library preparation and sequencing. ( A ) Gating strategy for FACS sorting of the indicated populations. ( B ) Principal components analysis of the top 300 differentially expressed genes by plotting contribution of each T cell population to the first two principal components (PC1 and PC2) (left). Gene set enrichment analysis (GSEA) of Foxp3 + pT regs versus “rest” within each intestinal compartment defined by the PC2 shows significant enrichment ( P

Techniques Used: Expressing, Mouse Assay, Purification, RNA Sequencing Assay, Sequencing, FACS

17) Product Images from "Limiting the priming dose of a SARS CoV-2 vaccine improves virus-specific immunity"

Article Title: Limiting the priming dose of a SARS CoV-2 vaccine improves virus-specific immunity

Journal: bioRxiv

doi: 10.1101/2021.03.31.437931

A LD/SD vaccine regimen elicits more functional CD8 T cell responses compared to a SD/SD vaccine regimen. In panels A-D, splenocytes were incubated with overlapping SARS CoV-2 peptide pools for 5 hr at 37°C in the presence of GolgiStop and GolgiPlug. (A) Representative FACS plots showing the frequencies of cytokine expressing SARS CoV-2-specific CD8 T cells. (B) Summary of SARS CoV-2-specific CD8 T cells that express the degranulation marker CD107a. (C) Summary of SARS CoV-2-specific CD8 T cells that express IFNγ. (D) Summary of SARS CoV-2-specific CD4 T cells that express IFNγ. (E) Representative FACS plots showing the frequencies of granzyme B and Ki67 expressing CD8 T cells. (F) Summary of Ki67 expression. (G) Summary of granzyme B expression. Panels E-G are gated on K b VL8+ cells (SARS CoV-2-specific). Data from panels F-G are indicated as mean fluorescence intensity (MFI). All data are from spleen. Data are from one experiment with n=4-5 per group. Experiment was repeated once with similar results. Indicated P values were determined by Mann-Whitney U test.
Figure Legend Snippet: A LD/SD vaccine regimen elicits more functional CD8 T cell responses compared to a SD/SD vaccine regimen. In panels A-D, splenocytes were incubated with overlapping SARS CoV-2 peptide pools for 5 hr at 37°C in the presence of GolgiStop and GolgiPlug. (A) Representative FACS plots showing the frequencies of cytokine expressing SARS CoV-2-specific CD8 T cells. (B) Summary of SARS CoV-2-specific CD8 T cells that express the degranulation marker CD107a. (C) Summary of SARS CoV-2-specific CD8 T cells that express IFNγ. (D) Summary of SARS CoV-2-specific CD4 T cells that express IFNγ. (E) Representative FACS plots showing the frequencies of granzyme B and Ki67 expressing CD8 T cells. (F) Summary of Ki67 expression. (G) Summary of granzyme B expression. Panels E-G are gated on K b VL8+ cells (SARS CoV-2-specific). Data from panels F-G are indicated as mean fluorescence intensity (MFI). All data are from spleen. Data are from one experiment with n=4-5 per group. Experiment was repeated once with similar results. Indicated P values were determined by Mann-Whitney U test.

Techniques Used: Functional Assay, Incubation, FACS, Expressing, Marker, Fluorescence, MANN-WHITNEY

Phenotypic validation of CD8 T cell responses after a single prime with the Ad5-SARS-2 spike vaccine. (A) Representative FACS plots showing the frequencies of SARS CoV-2-specific CD8 T cells (K b VL8) that differentiate into effector memory and central memory T cell subsets. (B) Summary of effector memory and central memory T cell subsets. (C) CD127 expression. (D) CD62L expression. (E) CD44 expression. (F) PD-1 expression. Panels B-F are gated from splenic SARS CoV-2-specific CD8 T cells (K b VL8). All data are from day 28 post-prime. Data are from one experiment with n=5 per group. Experiment was repeated two additional times with similar results. Indicated P values were determined by Mann-Whitney U test.
Figure Legend Snippet: Phenotypic validation of CD8 T cell responses after a single prime with the Ad5-SARS-2 spike vaccine. (A) Representative FACS plots showing the frequencies of SARS CoV-2-specific CD8 T cells (K b VL8) that differentiate into effector memory and central memory T cell subsets. (B) Summary of effector memory and central memory T cell subsets. (C) CD127 expression. (D) CD62L expression. (E) CD44 expression. (F) PD-1 expression. Panels B-F are gated from splenic SARS CoV-2-specific CD8 T cells (K b VL8). All data are from day 28 post-prime. Data are from one experiment with n=5 per group. Experiment was repeated two additional times with similar results. Indicated P values were determined by Mann-Whitney U test.

Techniques Used: FACS, Expressing, MANN-WHITNEY

Single cell RNA-seq analyses demonstrate that a low dose prime favors central memory CD8 T cell differentiation. Mice were immunized with 10 6 or 10 9 PFU of Ad5-SARS-2 spike, and at day 28, splenic CD8 T cells were MACS-sorted. Subsequently, live, CD8+, CD44+, K b VL8+ cells were FACS-sorted to ~99% purity for scRNA-seq. (A) UMAP plots showing populations colored by regimen (left plot). Standard and low dose cells were cluster separately and UMAP (right plot) shows unsupervised cell clusters. (B) Heatmap showing row-standardized expression of selected effector and memory genes (middle rows) or gene signatures (bottom rows). For each population, percentages of cells in each cluster are indicated (top row). (C) Violin plot showing the normalized expression of the Terminal Effector signature in the Standard and Low dose populations.
Figure Legend Snippet: Single cell RNA-seq analyses demonstrate that a low dose prime favors central memory CD8 T cell differentiation. Mice were immunized with 10 6 or 10 9 PFU of Ad5-SARS-2 spike, and at day 28, splenic CD8 T cells were MACS-sorted. Subsequently, live, CD8+, CD44+, K b VL8+ cells were FACS-sorted to ~99% purity for scRNA-seq. (A) UMAP plots showing populations colored by regimen (left plot). Standard and low dose cells were cluster separately and UMAP (right plot) shows unsupervised cell clusters. (B) Heatmap showing row-standardized expression of selected effector and memory genes (middle rows) or gene signatures (bottom rows). For each population, percentages of cells in each cluster are indicated (top row). (C) Violin plot showing the normalized expression of the Terminal Effector signature in the Standard and Low dose populations.

Techniques Used: RNA Sequencing Assay, Cell Differentiation, Mouse Assay, Magnetic Cell Separation, FACS, Expressing

Single cell TCR-seq analyses demonstrate that the prime dose does not alter public TCR clonotypes. Mice were primed with a LD (10 6 PFU) or a SD (10 9 PFU) of Ad5-SARS-2 spike, and at day 28, splenic CD8 T cells were MACS-sorted. Subsequently, live, CD8+, CD44+, K b VL8+ cells were FACS-sorted to ~99% purity for scTCR-Seq. Pie chart showing the distribution of TCRa and TCRb gene usage after SD prime (A) and LD prime (B). Total number above the pie chart show the number of single cells selected for the analyses, and different colors highlight the top 5 TCR usages and their relative proportion in each population.
Figure Legend Snippet: Single cell TCR-seq analyses demonstrate that the prime dose does not alter public TCR clonotypes. Mice were primed with a LD (10 6 PFU) or a SD (10 9 PFU) of Ad5-SARS-2 spike, and at day 28, splenic CD8 T cells were MACS-sorted. Subsequently, live, CD8+, CD44+, K b VL8+ cells were FACS-sorted to ~99% purity for scTCR-Seq. Pie chart showing the distribution of TCRa and TCRb gene usage after SD prime (A) and LD prime (B). Total number above the pie chart show the number of single cells selected for the analyses, and different colors highlight the top 5 TCR usages and their relative proportion in each population.

Techniques Used: Mouse Assay, Magnetic Cell Separation, FACS

A LD/SD vaccine regimen elicits superior CD8 T cells compared to a SD/SD vaccine regimen. (A) Experimental approach for evaluating how the priming dose of an Ad5-SARS-2 spike vaccine affects CD8 T cell responses in C57BL/6 mice. (B) Representative FACS plots showing the frequencies of SARS CoV-2-specific CD8 T cells (K b VL8+) in PBMCs. (C) Summary of SARS CoV-2-specific CD8 T cell responses in PBMCs. (D) Representative FACS plots showing the frequencies of SARS CoV-2-specific CD8 T cells (K b VL8+) in tissues. (E) Summary of SARS CoV-2-specific CD8 T cell responses in tissues. Data are from one experiment with n=5 per group. Experiment was repeated two additional times with similar results. Indicated P values were determined by Mann-Whitney U test. Error bars represent SEM.
Figure Legend Snippet: A LD/SD vaccine regimen elicits superior CD8 T cells compared to a SD/SD vaccine regimen. (A) Experimental approach for evaluating how the priming dose of an Ad5-SARS-2 spike vaccine affects CD8 T cell responses in C57BL/6 mice. (B) Representative FACS plots showing the frequencies of SARS CoV-2-specific CD8 T cells (K b VL8+) in PBMCs. (C) Summary of SARS CoV-2-specific CD8 T cell responses in PBMCs. (D) Representative FACS plots showing the frequencies of SARS CoV-2-specific CD8 T cells (K b VL8+) in tissues. (E) Summary of SARS CoV-2-specific CD8 T cell responses in tissues. Data are from one experiment with n=5 per group. Experiment was repeated two additional times with similar results. Indicated P values were determined by Mann-Whitney U test. Error bars represent SEM.

Techniques Used: Mouse Assay, FACS, MANN-WHITNEY

A LD prime elicits CD8 T cell responses with intrinsically superior anamnestic capacity. CD45.2+ mice were immunized intramuscularly with 10 6 or 10 9 PFU of Ad5-SARS-2 spike, and at day 28, splenic CD8 T cells were MACS-sorted. Subsequently, live, CD8+, CD44+, K b VL8+ cells were FACS-sorted to ~99% purity, and numbers were normalized for adoptive transfer into CD45.1+ recipient mice. (A) Experimental approach for evaluating secondary expansion of donor CD8 T cells. (B) Representative FACS plots showing the frequencies of donor CD8 T cells after boosting. (C) Summary of donor-derived CD8 T cells in PBMCs. (D) Summary of donor-derived CD8 T cells in spleen. (E) Summary of donor-derived CD8 T cells in draining lymph nodes. Data from panels B-E are from day 14 post-boost. Data are from one experiment with n=5 per group. Experiment was repeated once with similar results. Indicated P values were determined by Mann-Whitney U test.
Figure Legend Snippet: A LD prime elicits CD8 T cell responses with intrinsically superior anamnestic capacity. CD45.2+ mice were immunized intramuscularly with 10 6 or 10 9 PFU of Ad5-SARS-2 spike, and at day 28, splenic CD8 T cells were MACS-sorted. Subsequently, live, CD8+, CD44+, K b VL8+ cells were FACS-sorted to ~99% purity, and numbers were normalized for adoptive transfer into CD45.1+ recipient mice. (A) Experimental approach for evaluating secondary expansion of donor CD8 T cells. (B) Representative FACS plots showing the frequencies of donor CD8 T cells after boosting. (C) Summary of donor-derived CD8 T cells in PBMCs. (D) Summary of donor-derived CD8 T cells in spleen. (E) Summary of donor-derived CD8 T cells in draining lymph nodes. Data from panels B-E are from day 14 post-boost. Data are from one experiment with n=5 per group. Experiment was repeated once with similar results. Indicated P values were determined by Mann-Whitney U test.

Techniques Used: Mouse Assay, Magnetic Cell Separation, FACS, Adoptive Transfer Assay, Derivative Assay, MANN-WHITNEY

18) Product Images from "Microbial Energy Metabolism Fuels a CSF2-dependent Intestinal Macrophage Niche within Tertiary Lymphoid Organs"

Article Title: Microbial Energy Metabolism Fuels a CSF2-dependent Intestinal Macrophage Niche within Tertiary Lymphoid Organs

Journal: bioRxiv

doi: 10.1101/2022.03.23.485563

CSF2-producing ILC3s in TLOs induce CCR2 + Tim-4 - CD4 - MPs. (A) Representative live image of a colonic tertiary lymphoid organ (TLO) in Rorc +/EGFP Ccr2 +/RFP mice. (B) Representative immunofluorescence images of colonic lamina propria (LP) and TLOs of Cx3cr1 +/GFP Ccr2 +/RFP mice stained for Tim-4 and DNA. (C) Proportion of CCR2-RFP + of CX3CR1-GFP + cells based on CellProfiler quantification of images in each colonic LP (CLP) region and TLOs, as indicated. (D) Colonic MP composition in adult sex-matched littermate mice as indicated. (E) 10 4 FACS-sorted small intestinal ILC3s from either WT or Csf2 -/- mice were adoptively transferred into Rag2 -/- Il2r -/- mice, and recipients were analyzed at 6 weeks (left). Quantifications (right) of colonic MP populations post-transfer, as indicated. Data shown are representative of at least three independent experiments with at least three mice per group per experiment. One-way ANOVA with post-hoc Tukey’s test was performed for (C) and (E); *p
Figure Legend Snippet: CSF2-producing ILC3s in TLOs induce CCR2 + Tim-4 - CD4 - MPs. (A) Representative live image of a colonic tertiary lymphoid organ (TLO) in Rorc +/EGFP Ccr2 +/RFP mice. (B) Representative immunofluorescence images of colonic lamina propria (LP) and TLOs of Cx3cr1 +/GFP Ccr2 +/RFP mice stained for Tim-4 and DNA. (C) Proportion of CCR2-RFP + of CX3CR1-GFP + cells based on CellProfiler quantification of images in each colonic LP (CLP) region and TLOs, as indicated. (D) Colonic MP composition in adult sex-matched littermate mice as indicated. (E) 10 4 FACS-sorted small intestinal ILC3s from either WT or Csf2 -/- mice were adoptively transferred into Rag2 -/- Il2r -/- mice, and recipients were analyzed at 6 weeks (left). Quantifications (right) of colonic MP populations post-transfer, as indicated. Data shown are representative of at least three independent experiments with at least three mice per group per experiment. One-way ANOVA with post-hoc Tukey’s test was performed for (C) and (E); *p

Techniques Used: Mouse Assay, Immunofluorescence, Staining, FACS

19) Product Images from "RORγt+ cells selectively express redundant cation channels linked to the Golgi apparatus"

Article Title: RORγt+ cells selectively express redundant cation channels linked to the Golgi apparatus

Journal: Scientific Reports

doi: 10.1038/srep23682

Tmem176a and b mRNA expression in intestinal ILC3s. ( a ) Lymphocytes from intestinal lamina propria of RORγt-fate map mice ( Rorc ( γt )- Cre TG × Rosa26-tdRFP ) were isolated. Lineage-negative (CD11b − CD11c − CD19 − TCRαβ − TCRγδ − ) RFP + NK1.1 − ILC3-enriched and RFP − NK1.1 + ILC1s were FACS-sorted. In parallel, CD11b/c + and lineage-negative NK1.1 + conventional NK/ILC1s were FACS-sorted from the spleen. ( b ) Expression of indicated genes (mean ± SD) was assessed by quantitative RT-PCR in each population isolated from 3–4 independent mice. Statistically significant differences between ILC3s and the other populations are indicated: *p
Figure Legend Snippet: Tmem176a and b mRNA expression in intestinal ILC3s. ( a ) Lymphocytes from intestinal lamina propria of RORγt-fate map mice ( Rorc ( γt )- Cre TG × Rosa26-tdRFP ) were isolated. Lineage-negative (CD11b − CD11c − CD19 − TCRαβ − TCRγδ − ) RFP + NK1.1 − ILC3-enriched and RFP − NK1.1 + ILC1s were FACS-sorted. In parallel, CD11b/c + and lineage-negative NK1.1 + conventional NK/ILC1s were FACS-sorted from the spleen. ( b ) Expression of indicated genes (mean ± SD) was assessed by quantitative RT-PCR in each population isolated from 3–4 independent mice. Statistically significant differences between ILC3s and the other populations are indicated: *p

Techniques Used: Expressing, Mouse Assay, Isolation, FACS, Quantitative RT-PCR

20) Product Images from "PD-1 blockade restores helper activity of tumor-infiltrating, exhausted PD-1hiCD39+ CD4 T cells"

Article Title: PD-1 blockade restores helper activity of tumor-infiltrating, exhausted PD-1hiCD39+ CD4 T cells

Journal: JCI Insight

doi: 10.1172/jci.insight.142513

PD-1 hi CD39 + CD4 Tconv TILs encompass tumor Ag–specific cells and respond to PD-1 blockade by enhancing DC-mediated CD8 T cell proliferation. ( A – C ) Ex vivo isolated CD4 + TILs from one OC NY-ESO-1–seropositive patient were FACS-sorted into PD-1 – CD39 – , PD-1 hi CD39 – , and PD-1 hi CD39 + CD4 Tconv (CD3 + CD4 + CD25 - CD127 + ) subsets and cloned. ( A and B ) Clonal populations were stained and analyzed by flow cytometry. ( A ) PD-1 versus CD39 expression in clones representative of the 3 sorted Tconv populations. ( B ) Proportions of PD-1 + and CD39 + cells are summarized for all clones derived from PD-1 – CD39 – ( n = 54), PD-1 hi CD39 – ( n = 17), and PD-1 hi CD39 + ( n = 22) subsets. ( C ) IFN-γ concentration in the supernatant was quantified by ELISA for each clone stimulated with NY-ESO-1 peptide pool (fold increase over unstimulated condition) ( n as in B ). ( D – F ) Ex vivo CD4 + TILs (± anti–PD-1 mAbs pretreatment) were cocultured with iDCs in the presence or absence of PHA. ( D ) TNF-α, IFN-γ, IL-2, and IL-12 concentrations were quantified by CBA in the 24-hour supernatants ( n = 3). ( E ) Histogram plots show CD154 expression in CD4 Tconvs after 6-hours stimulation. Proportions of CD154 + cells are summarized ( n = 5). ( F ) Histogram plots show CD86 expression in DCs in day 2 cultures. MFI of CD86 staining are summarized ( n = 4). ( G ) CD4 + TILs from OC NY-ESO-1–seropositive patients (± anti–PD-1 mAbs pretreatment); autologous iDCs and circulating CD8 T cells were cocultured in the presence of NY-ESO-1 peptides, stained with MHC-I/NY-ESO-1 peptide multimers on day 10, and analyzed by flow cytometry. Examples of dot plots show multimer staining and CD8 expression and proportions of multimer + CD8 + cells are summarized ( n = 5). A 2-tailed paired t test was used to compare variables ( E and F ). Bonferroni’s correction was applied to account for multiple testing ( E and F ) and significance level was adjusted accordingly (* P
Figure Legend Snippet: PD-1 hi CD39 + CD4 Tconv TILs encompass tumor Ag–specific cells and respond to PD-1 blockade by enhancing DC-mediated CD8 T cell proliferation. ( A – C ) Ex vivo isolated CD4 + TILs from one OC NY-ESO-1–seropositive patient were FACS-sorted into PD-1 – CD39 – , PD-1 hi CD39 – , and PD-1 hi CD39 + CD4 Tconv (CD3 + CD4 + CD25 - CD127 + ) subsets and cloned. ( A and B ) Clonal populations were stained and analyzed by flow cytometry. ( A ) PD-1 versus CD39 expression in clones representative of the 3 sorted Tconv populations. ( B ) Proportions of PD-1 + and CD39 + cells are summarized for all clones derived from PD-1 – CD39 – ( n = 54), PD-1 hi CD39 – ( n = 17), and PD-1 hi CD39 + ( n = 22) subsets. ( C ) IFN-γ concentration in the supernatant was quantified by ELISA for each clone stimulated with NY-ESO-1 peptide pool (fold increase over unstimulated condition) ( n as in B ). ( D – F ) Ex vivo CD4 + TILs (± anti–PD-1 mAbs pretreatment) were cocultured with iDCs in the presence or absence of PHA. ( D ) TNF-α, IFN-γ, IL-2, and IL-12 concentrations were quantified by CBA in the 24-hour supernatants ( n = 3). ( E ) Histogram plots show CD154 expression in CD4 Tconvs after 6-hours stimulation. Proportions of CD154 + cells are summarized ( n = 5). ( F ) Histogram plots show CD86 expression in DCs in day 2 cultures. MFI of CD86 staining are summarized ( n = 4). ( G ) CD4 + TILs from OC NY-ESO-1–seropositive patients (± anti–PD-1 mAbs pretreatment); autologous iDCs and circulating CD8 T cells were cocultured in the presence of NY-ESO-1 peptides, stained with MHC-I/NY-ESO-1 peptide multimers on day 10, and analyzed by flow cytometry. Examples of dot plots show multimer staining and CD8 expression and proportions of multimer + CD8 + cells are summarized ( n = 5). A 2-tailed paired t test was used to compare variables ( E and F ). Bonferroni’s correction was applied to account for multiple testing ( E and F ) and significance level was adjusted accordingly (* P

Techniques Used: Ex Vivo, Isolation, FACS, Clone Assay, Staining, Flow Cytometry, Expressing, Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Crocin Bleaching Assay

21) Product Images from "Anti-apoptotic ARC protein confers chemoresistance by controlling leukemia-microenvironment interactions through a NFκB/IL1β signaling network"

Article Title: Anti-apoptotic ARC protein confers chemoresistance by controlling leukemia-microenvironment interactions through a NFκB/IL1β signaling network

Journal: Oncotarget

doi: 10.18632/oncotarget.7911

MSCs induce the expression of IL1β in AML cells OCI-AML3 cells were cultured alone or co-cultured with ARC KD or control MSCs for 48 h. CD45 + CD90 − AML cells were FACS-sorted from the floaters (collected from cells in the suspension and after PBS wash) and attached (collected by trypsinization) cells. IL1β RNA levels were determined in sorted OCI-AML3 cells and the cells cultured alone by real-time RT-PCR.
Figure Legend Snippet: MSCs induce the expression of IL1β in AML cells OCI-AML3 cells were cultured alone or co-cultured with ARC KD or control MSCs for 48 h. CD45 + CD90 − AML cells were FACS-sorted from the floaters (collected from cells in the suspension and after PBS wash) and attached (collected by trypsinization) cells. IL1β RNA levels were determined in sorted OCI-AML3 cells and the cells cultured alone by real-time RT-PCR.

Techniques Used: Expressing, Cell Culture, FACS, Quantitative RT-PCR

ARC in AML modulates CCL2, CCL4, and CXCL12 expression in MSCs ( A ) MSCs were cultured alone or with ARC KD OCI-AML3, ARC OE KG-1, or the respective control cells for 48 h and MSCs were FACS sorted conservatively as marked in the boxes for CD45 − CD90 + cells. ( B ) CCL2, CCL4, and CXCL12 levels in MSCs were determined by quantitative RT-PCR.
Figure Legend Snippet: ARC in AML modulates CCL2, CCL4, and CXCL12 expression in MSCs ( A ) MSCs were cultured alone or with ARC KD OCI-AML3, ARC OE KG-1, or the respective control cells for 48 h and MSCs were FACS sorted conservatively as marked in the boxes for CD45 − CD90 + cells. ( B ) CCL2, CCL4, and CXCL12 levels in MSCs were determined by quantitative RT-PCR.

Techniques Used: Expressing, Cell Culture, FACS, Quantitative RT-PCR

22) Product Images from " Role of IFN-? in induction of Foxp3 and conversion of CD4+ CD25– T cells to CD4+ Tregs"

Article Title: Role of IFN-? in induction of Foxp3 and conversion of CD4+ CD25– T cells to CD4+ Tregs

Journal: Journal of Clinical Investigation

doi: 10.1172/JCI25826

Induction of Foxp3 expression and regulatory properties by IFN-γ in human cells. ( A ) PBMC preparations were obtained from 10 healthy individuals and cultured in the presence or absence of the indicated cytokines (25 ng/ml) for 24 hours. CD4 + CD25 + Tregs were used as a reference. The resulting cells were analyzed for the expression of Foxp3 by real-time PCR and immunoblot. The real-time PCR histogram represents analysis of 10 individual specimens. Relative change in Foxp3 expression in immunoblot is presented in folds (intensity of experimental band/intensity of control band). ( B ) Purified CD4 + CD25 – T cell preparations ( n = 10) were cultured in the presence of IFN-γ at the indicated concentrations for 24 hours and measured for Foxp3 expression. ( C ) CD4 + CD25 – T cells treated in the presence or absence of IFN-γ (25 ng/ml) were analyzed for intracellular Foxp3 expression by flow cytometry. CD4 + CD25 + Tregs were used as a reference. ( D ) CD4 + CD25 – T cells were cultured in the presence of IFN-γ and the indicated antibodies (10 μg/ml). The resulting T cells were analyzed for mRNA expression of Foxp3 by real-time PCR. Horizontal line represents the level of Foxp3 expression in untreated CD4 + CD25 – T cells. ( E ) Purified CD4 + CD25 – T cells were treated with IFN-γ under the experimental conditions described above. The resulting T cells were FACS sorted and assayed for inhibitory activity on the proliferation of autologous CD4 + CD25 – T cells. Results are expressed as mean percentage inhibition ± SEM from 5 independent experiments. Asterisks indicate that differences between groups are statistically significant; * P
Figure Legend Snippet: Induction of Foxp3 expression and regulatory properties by IFN-γ in human cells. ( A ) PBMC preparations were obtained from 10 healthy individuals and cultured in the presence or absence of the indicated cytokines (25 ng/ml) for 24 hours. CD4 + CD25 + Tregs were used as a reference. The resulting cells were analyzed for the expression of Foxp3 by real-time PCR and immunoblot. The real-time PCR histogram represents analysis of 10 individual specimens. Relative change in Foxp3 expression in immunoblot is presented in folds (intensity of experimental band/intensity of control band). ( B ) Purified CD4 + CD25 – T cell preparations ( n = 10) were cultured in the presence of IFN-γ at the indicated concentrations for 24 hours and measured for Foxp3 expression. ( C ) CD4 + CD25 – T cells treated in the presence or absence of IFN-γ (25 ng/ml) were analyzed for intracellular Foxp3 expression by flow cytometry. CD4 + CD25 + Tregs were used as a reference. ( D ) CD4 + CD25 – T cells were cultured in the presence of IFN-γ and the indicated antibodies (10 μg/ml). The resulting T cells were analyzed for mRNA expression of Foxp3 by real-time PCR. Horizontal line represents the level of Foxp3 expression in untreated CD4 + CD25 – T cells. ( E ) Purified CD4 + CD25 – T cells were treated with IFN-γ under the experimental conditions described above. The resulting T cells were FACS sorted and assayed for inhibitory activity on the proliferation of autologous CD4 + CD25 – T cells. Results are expressed as mean percentage inhibition ± SEM from 5 independent experiments. Asterisks indicate that differences between groups are statistically significant; * P

Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Purification, Flow Cytometry, Cytometry, FACS, Activity Assay, Inhibition

23) Product Images from "NKG2A and HLA-E define a novel alternative immune checkpoint axis in bladder cancer"

Article Title: NKG2A and HLA-E define a novel alternative immune checkpoint axis in bladder cancer

Journal: bioRxiv

doi: 10.1101/2022.03.04.482960

TCR-independent function is acquired by CD8 T cells upon NKG2A acquisition and is enhanced with NKG2A blockade in bladder cancer. (A-C) CD8 T cells were isolated from bladder tumor-draining lymph nodes and expanded for 8-13 days with IL-2, IL-7, IL-15 and CD3/CD28 tetramer. NKG2A − PD1 − cells were then FACS-sorted and expanded during 3 additional days with or without TGF-β, prior to co-culture with K562. CyTOF was performed at all timepoints. (A) NKG2A/PD1 expression on CD49a − NKG2A − PD1 − CD8 T cells after 3-day culture with or without TGF-β (n=5). (B) Upregulation of functional markers upon K562 stimulation of CD49a − and CD49a + NKG2A − PD1 − CD8 T cells after 3-day expansion in the presence of TGF-β (n=5 and 6, respectively). (C) Phenotype and response to K562 following 3-day expansion of NKG2A − PD1 − CD8 T cells and NKG2A/PD1 upregulation (n=11). (D-E) CD8 T cells were isolated from bladder tumors (n=6) and expanded for 11-17 days with IL-2, IL-7, IL-15 and CD3/CD28 tetramer, prior to co-culture with HLA-E + K562. (D) Effect of NKG2A blockade (monalizumab) on CD8 T cell degranulation upon HLA-E + K562 stimulation. (E) Spearman correlation between HLA-E expression on CD45 − bladder tumor cells and frequency of CD107a expression by NKG2A + CD8 T cells that is restored by monalizumab upon co-culture with HLA-E + K562. Paired t-tests were used in (B), (C), (D) and Spearman correlation in (E). Corrections for multiple comparisons were applied in (B) and (C). Only the significant observations (p
Figure Legend Snippet: TCR-independent function is acquired by CD8 T cells upon NKG2A acquisition and is enhanced with NKG2A blockade in bladder cancer. (A-C) CD8 T cells were isolated from bladder tumor-draining lymph nodes and expanded for 8-13 days with IL-2, IL-7, IL-15 and CD3/CD28 tetramer. NKG2A − PD1 − cells were then FACS-sorted and expanded during 3 additional days with or without TGF-β, prior to co-culture with K562. CyTOF was performed at all timepoints. (A) NKG2A/PD1 expression on CD49a − NKG2A − PD1 − CD8 T cells after 3-day culture with or without TGF-β (n=5). (B) Upregulation of functional markers upon K562 stimulation of CD49a − and CD49a + NKG2A − PD1 − CD8 T cells after 3-day expansion in the presence of TGF-β (n=5 and 6, respectively). (C) Phenotype and response to K562 following 3-day expansion of NKG2A − PD1 − CD8 T cells and NKG2A/PD1 upregulation (n=11). (D-E) CD8 T cells were isolated from bladder tumors (n=6) and expanded for 11-17 days with IL-2, IL-7, IL-15 and CD3/CD28 tetramer, prior to co-culture with HLA-E + K562. (D) Effect of NKG2A blockade (monalizumab) on CD8 T cell degranulation upon HLA-E + K562 stimulation. (E) Spearman correlation between HLA-E expression on CD45 − bladder tumor cells and frequency of CD107a expression by NKG2A + CD8 T cells that is restored by monalizumab upon co-culture with HLA-E + K562. Paired t-tests were used in (B), (C), (D) and Spearman correlation in (E). Corrections for multiple comparisons were applied in (B) and (C). Only the significant observations (p

Techniques Used: Isolation, FACS, Co-Culture Assay, Expressing, Functional Assay

24) Product Images from "Synergistic Interactions between HDAC and Sirtuin Inhibitors in Human Leukemia Cells"

Article Title: Synergistic Interactions between HDAC and Sirtuin Inhibitors in Human Leukemia Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0022739

HDAC inhibitor-induced Bax upregulation contributes to the synergy with sirtuin inhibitors. A, 3×10 6 primary AML cells/well were plated in 6-well plates and incubated in the presence or absence of 50 µM cambinol, 100 µg/ml VA, or their combination. ΔΨ m was monitored at the indicated time points by TMRE staining and flow cytometry. B, 1×10 6 Jurkat cells were plated in 6-well plates and incubated for 48 h in the presence or absence of 100 µg/ml VA. Thereafter, intracellular Bax content was determined by flow cytometry. C, D, Jurkat cells were transduced with either pMIG or pMIG-Bax. Infected cells were FACS sorted, allowed to expand, and subsequently used for flow cytometric detection of intracellular Bax (C) and for viability assays (D). For these, pMIG- or pMIG-Bax-transduced Jurkat were plated in 96-well plates and incubated in the presence or absence of EX527 or cambinol at the indicated concentrations. Viability was determined by PI staining and flow cytometry 48 h later. A–C, one representative experiment out of three is presented. D, Results are means ± SD of three separate experiments.
Figure Legend Snippet: HDAC inhibitor-induced Bax upregulation contributes to the synergy with sirtuin inhibitors. A, 3×10 6 primary AML cells/well were plated in 6-well plates and incubated in the presence or absence of 50 µM cambinol, 100 µg/ml VA, or their combination. ΔΨ m was monitored at the indicated time points by TMRE staining and flow cytometry. B, 1×10 6 Jurkat cells were plated in 6-well plates and incubated for 48 h in the presence or absence of 100 µg/ml VA. Thereafter, intracellular Bax content was determined by flow cytometry. C, D, Jurkat cells were transduced with either pMIG or pMIG-Bax. Infected cells were FACS sorted, allowed to expand, and subsequently used for flow cytometric detection of intracellular Bax (C) and for viability assays (D). For these, pMIG- or pMIG-Bax-transduced Jurkat were plated in 96-well plates and incubated in the presence or absence of EX527 or cambinol at the indicated concentrations. Viability was determined by PI staining and flow cytometry 48 h later. A–C, one representative experiment out of three is presented. D, Results are means ± SD of three separate experiments.

Techniques Used: Incubation, Staining, Flow Cytometry, Cytometry, Transduction, Infection, FACS

25) Product Images from "Stability and function of regulatory T cells expressing the transcription factor T-bet"

Article Title: Stability and function of regulatory T cells expressing the transcription factor T-bet

Journal: Nature

doi: 10.1038/nature22360

Fate mapping T-bet-expressing Treg cells during infectious challenge a , Preferential expansion of CD44 hi RFP − vs. CD44 hi RFP + CD4 effector T cells during Nippostrongylus brasiliensis ( Nb ) infection. Flow cytometry analysis of splenic (above) and lung (below) CD4 + Thy1.1 − cells from PBS- (left) and Nb - (right) challenged mice. b , Flow cytometry of splenic CD4 + Thy1.1 + (left) and Thy1.1 − (right) cells of PBS- (above) and Lm- (below) challenged mice, as indicated in Fig 2a . Numbers indicate percent RFP + (left) and YFP + (right) cells. c , (Above) schematic of experiment. CD44 lo CD62L hi RFP − , CD44 hi RFP − , and CD44 hi RFP hi CXCR3 hi CD4 + Thy1.1 + cells were FACS-sorted from pooled spleens and lymph nodes of Tbx21 RFP-CreERT2 mice and transferred into lymphoreplete hosts one day before PBS or Lm challenge. (Below) Flow cytometry of transferred populations (indicated on left) on d9 in spleens of PBS- (left) or Lm - (right) challenged hosts. d , Representative histograms of RFP and CXCR3 expression on total CD4 + Thy1.1 + (shaded histograms) or Th1.1 + YFP + (open histograms) cells from spleens of PBS- (black) or Lm - (red) challenged mice, as indicted in Fig 2a . e–g, eGFP expression in PBS or Lm challenged Tbx21 RFP-CreERT2 IL-10 eGFP mice. e , Schematic of tamoxifen (tx) administration to Tbx21 RFP-CreERT2 IL10 eGFP/WT mice for data shown in ( f,g ). f , Flow cytometry of Treg (above) and YFP + Treg (below) cells in spleens of PBS (left) and Lm (right) treated mice. g , (Left) percent RFP - eGFP + and RFP + eGFP + among Treg cells, as gated in ( f, above); (right) percent eGFP + cells among YFP + Treg cells, as gated in ( f , below). h , Schematic of Lm reinfection in Tbx21 RFP-CreERT2 IL10 eGFP/WT mice for data shown in ( i,j ); 1 o and 2 o indicate primary and secondary challenge, respectively. i , Flow cytometry of cells in Tbx21 RFP-CreERT2 IL-10 eGFP mice on d65, treated as indicated above. j , Percent RFP − eGFP + and RFP + eGFP + cells among Thy1.1 + cells, as gated in ( i ). All data are representative of ≥ 2 experiments, n≥ 2 mice per group each. Bars, mean±s.e.m. Two-tailed t test (NS – not significant).
Figure Legend Snippet: Fate mapping T-bet-expressing Treg cells during infectious challenge a , Preferential expansion of CD44 hi RFP − vs. CD44 hi RFP + CD4 effector T cells during Nippostrongylus brasiliensis ( Nb ) infection. Flow cytometry analysis of splenic (above) and lung (below) CD4 + Thy1.1 − cells from PBS- (left) and Nb - (right) challenged mice. b , Flow cytometry of splenic CD4 + Thy1.1 + (left) and Thy1.1 − (right) cells of PBS- (above) and Lm- (below) challenged mice, as indicated in Fig 2a . Numbers indicate percent RFP + (left) and YFP + (right) cells. c , (Above) schematic of experiment. CD44 lo CD62L hi RFP − , CD44 hi RFP − , and CD44 hi RFP hi CXCR3 hi CD4 + Thy1.1 + cells were FACS-sorted from pooled spleens and lymph nodes of Tbx21 RFP-CreERT2 mice and transferred into lymphoreplete hosts one day before PBS or Lm challenge. (Below) Flow cytometry of transferred populations (indicated on left) on d9 in spleens of PBS- (left) or Lm - (right) challenged hosts. d , Representative histograms of RFP and CXCR3 expression on total CD4 + Thy1.1 + (shaded histograms) or Th1.1 + YFP + (open histograms) cells from spleens of PBS- (black) or Lm - (red) challenged mice, as indicted in Fig 2a . e–g, eGFP expression in PBS or Lm challenged Tbx21 RFP-CreERT2 IL-10 eGFP mice. e , Schematic of tamoxifen (tx) administration to Tbx21 RFP-CreERT2 IL10 eGFP/WT mice for data shown in ( f,g ). f , Flow cytometry of Treg (above) and YFP + Treg (below) cells in spleens of PBS (left) and Lm (right) treated mice. g , (Left) percent RFP - eGFP + and RFP + eGFP + among Treg cells, as gated in ( f, above); (right) percent eGFP + cells among YFP + Treg cells, as gated in ( f , below). h , Schematic of Lm reinfection in Tbx21 RFP-CreERT2 IL10 eGFP/WT mice for data shown in ( i,j ); 1 o and 2 o indicate primary and secondary challenge, respectively. i , Flow cytometry of cells in Tbx21 RFP-CreERT2 IL-10 eGFP mice on d65, treated as indicated above. j , Percent RFP − eGFP + and RFP + eGFP + cells among Thy1.1 + cells, as gated in ( i ). All data are representative of ≥ 2 experiments, n≥ 2 mice per group each. Bars, mean±s.e.m. Two-tailed t test (NS – not significant).

Techniques Used: Expressing, Infection, Flow Cytometry, Cytometry, Mouse Assay, FACS, Two Tailed Test

26) Product Images from "Allergen recognition by specific effector Th2 cells enables IL-2-dependent activation of regulatory T cell responses in humans"

Article Title: Allergen recognition by specific effector Th2 cells enables IL-2-dependent activation of regulatory T cell responses in humans

Journal: medRxiv

doi: 10.1101/2022.05.17.22275017

T cell responses to peanut allergens are associated with the activation of unique subsets of highly differentiated effector Th2 cells and memory regulatory T cells (Tregs) in peanut allergic (PA) subjects. A . Overlap of the activated populations after stimulation with crude peanut extract (CPE) for 6h, 24h, and 48h represented by Venn diagrams (n=24-30). Numbers inside the circles indicate the percentage (± SEM) of non-overlapped cells and numbers next to the lines specify the overlap percentage (± SEM). Different colors designate different activated populations. B . Estimated Chao1 alpha diversity of TCRβ repertoire in activated populations and total memory CD4 + T cells sorted from PA subjects stimulated with CPE for 6h and 24h (n=3). C . Percentage of the mature effector ( m T EM ), effector (T EM ), and central (T CM ) memory CD4 + T cells in the activated populations after stimulation of PBMCs from PA subjects with CPE for 6h, 24h, and 48h (n=25). D . Percentage of chemokine receptor co-expression in activated populations after CPE-stimulation for 6h, 24h, and 48h (n=5). E . Heatmaps of the percentage of expression for each marker in Foxp3 - T cells (left) and Tregs (Foxp3 + , right) in activated populations after CPE-stimulation for 6h, 24h, and 48h (n=5). In (B) and (C) each data point is one individual (mean ± SEM) and bar graphs represent mean + SEM in (D). Statistical analyses by mixed-effect analysis with Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test in (B) and (C). In (D), dashed line represents cut off above populations are significantly increased (mixed-effect analysis accounting for subject levels repeated measures at the three timepoints studied with Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test, p
Figure Legend Snippet: T cell responses to peanut allergens are associated with the activation of unique subsets of highly differentiated effector Th2 cells and memory regulatory T cells (Tregs) in peanut allergic (PA) subjects. A . Overlap of the activated populations after stimulation with crude peanut extract (CPE) for 6h, 24h, and 48h represented by Venn diagrams (n=24-30). Numbers inside the circles indicate the percentage (± SEM) of non-overlapped cells and numbers next to the lines specify the overlap percentage (± SEM). Different colors designate different activated populations. B . Estimated Chao1 alpha diversity of TCRβ repertoire in activated populations and total memory CD4 + T cells sorted from PA subjects stimulated with CPE for 6h and 24h (n=3). C . Percentage of the mature effector ( m T EM ), effector (T EM ), and central (T CM ) memory CD4 + T cells in the activated populations after stimulation of PBMCs from PA subjects with CPE for 6h, 24h, and 48h (n=25). D . Percentage of chemokine receptor co-expression in activated populations after CPE-stimulation for 6h, 24h, and 48h (n=5). E . Heatmaps of the percentage of expression for each marker in Foxp3 - T cells (left) and Tregs (Foxp3 + , right) in activated populations after CPE-stimulation for 6h, 24h, and 48h (n=5). In (B) and (C) each data point is one individual (mean ± SEM) and bar graphs represent mean + SEM in (D). Statistical analyses by mixed-effect analysis with Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test in (B) and (C). In (D), dashed line represents cut off above populations are significantly increased (mixed-effect analysis accounting for subject levels repeated measures at the three timepoints studied with Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test, p

Techniques Used: Activation Assay, Expressing, Marker

Peanut allergens induce an early T cell response driven by CD154 + CD69 + cells and a delayed activation of suppressive regulatory T cells (Tregs) in peanut allergic (PA) subjects. A . Minimal spanning tree visualization of FlowSOM clustering analysis of memory (CD45RA) - CD4 + T cells of PA subjects stimulated with crude peanut extract (CPE) for 6h, 24h, and 48h (n=5). Different nodes indicate the relative size of the cluster identified. Meta-clusters (MC) are indicated with different numbers. B . Intensity of expression of activation markers visualized by a star chart in each node of the FlowSOM clustering analysis (n=5). Each pie height indicates intensity of expression. C . Percentage of the activated populations identified in memory CD4 + T cells unstimulated (CTRL) or stimulated with CPE for 6h, 24h, and 48h (n=30). D . Percentage of Foxp3 - (white) and Foxp3 + (green) CD4 + T cells in the activated populations after CPE-stimulation for 6h, 24h, and 48h (n=13-14). E . Inhibition percentage of CD25 - CD4 + T cell proliferation induced by activated Tregs populations sorted from polyclonally activated PBMCs. Suppression of CD25 - CD4 + T cell proliferation was calculated via CFSE dilution. F . Quantification of IL-4 and IL-5 released by polarized Th2 cells alone or cultured with activated Treg populations sorted from polyclonally activated PBMCs for 72h. In (C) each data point is one individual (mean ± SEM), each point represents the mean ± SEM of three independent experiments performed in triplicate in (E), and bar graphs represent mean + SEM of three independent experiments performed in triplicate in (F). Statistical analyses by mixed-effect analysis with Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test in (C) and (E), and ordinary one-way ANOVA with Geisser-Greenhouse correction followed by Dunnett’s multiple comparisons test in (F). *P
Figure Legend Snippet: Peanut allergens induce an early T cell response driven by CD154 + CD69 + cells and a delayed activation of suppressive regulatory T cells (Tregs) in peanut allergic (PA) subjects. A . Minimal spanning tree visualization of FlowSOM clustering analysis of memory (CD45RA) - CD4 + T cells of PA subjects stimulated with crude peanut extract (CPE) for 6h, 24h, and 48h (n=5). Different nodes indicate the relative size of the cluster identified. Meta-clusters (MC) are indicated with different numbers. B . Intensity of expression of activation markers visualized by a star chart in each node of the FlowSOM clustering analysis (n=5). Each pie height indicates intensity of expression. C . Percentage of the activated populations identified in memory CD4 + T cells unstimulated (CTRL) or stimulated with CPE for 6h, 24h, and 48h (n=30). D . Percentage of Foxp3 - (white) and Foxp3 + (green) CD4 + T cells in the activated populations after CPE-stimulation for 6h, 24h, and 48h (n=13-14). E . Inhibition percentage of CD25 - CD4 + T cell proliferation induced by activated Tregs populations sorted from polyclonally activated PBMCs. Suppression of CD25 - CD4 + T cell proliferation was calculated via CFSE dilution. F . Quantification of IL-4 and IL-5 released by polarized Th2 cells alone or cultured with activated Treg populations sorted from polyclonally activated PBMCs for 72h. In (C) each data point is one individual (mean ± SEM), each point represents the mean ± SEM of three independent experiments performed in triplicate in (E), and bar graphs represent mean + SEM of three independent experiments performed in triplicate in (F). Statistical analyses by mixed-effect analysis with Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test in (C) and (E), and ordinary one-way ANOVA with Geisser-Greenhouse correction followed by Dunnett’s multiple comparisons test in (F). *P

Techniques Used: Activation Assay, Expressing, Inhibition, Cell Culture

27) Product Images from "Alternative RNA splicing in the endothelium mediated in part by Rbfox2 regulates the arterial response to low flow"

Article Title: Alternative RNA splicing in the endothelium mediated in part by Rbfox2 regulates the arterial response to low flow

Journal: eLife

doi: 10.7554/eLife.29494

Enrichment of endothelial RNA demonstrated by lineage markers in mT/mG mice. The mT/mG mice constitutively express tdTomato in all cells from a CMV enhancer/chicken beta-actin core promoter (pCA). Upon Cre activity, in this case driven by the endothelial-specific Cdh5(PAC)-CreERT2 , the tdTomato is excised, and a downstream eGFP is expressed instead. Since tdTomato is expressed in all non-endothelial cells and eGFP is expressed in all endothelial cells, the ratio may be used as an indicator of endothelial to non-endothelial mRNA levels in the isolate. The comparison of RNA-seq reads assigned to eGFP or tdTomato from Cdh5(PAC)-CreERT2; Rbfox2 ff; mT/mG (N = 2 pools) mice or Rbfox2 ff; mT/mG mice (N = 2 pools) is shown. Cultured and FACs-purified eGFP +cells from Cdh5(PAC)-CreERT2; Rbfox2 ff; mT/mG mouse aorta is also shown (N = 1).
Figure Legend Snippet: Enrichment of endothelial RNA demonstrated by lineage markers in mT/mG mice. The mT/mG mice constitutively express tdTomato in all cells from a CMV enhancer/chicken beta-actin core promoter (pCA). Upon Cre activity, in this case driven by the endothelial-specific Cdh5(PAC)-CreERT2 , the tdTomato is excised, and a downstream eGFP is expressed instead. Since tdTomato is expressed in all non-endothelial cells and eGFP is expressed in all endothelial cells, the ratio may be used as an indicator of endothelial to non-endothelial mRNA levels in the isolate. The comparison of RNA-seq reads assigned to eGFP or tdTomato from Cdh5(PAC)-CreERT2; Rbfox2 ff; mT/mG (N = 2 pools) mice or Rbfox2 ff; mT/mG mice (N = 2 pools) is shown. Cultured and FACs-purified eGFP +cells from Cdh5(PAC)-CreERT2; Rbfox2 ff; mT/mG mouse aorta is also shown (N = 1).

Techniques Used: Mouse Assay, Activity Assay, RNA Sequencing Assay, Cell Culture, FACS, Purification

Arterial cells cultured in vitro replicate splicing changes observed under low and disturbed flow in vivo. Comparison of the change in inclusion frequency by MISO for individual splicing events of the classes shown (by color code on bottom), relative to high-flow arterial endothelium in vivo. Other splice events include A3SS, A5SS, MXE and RI. Only flow-regulated alternative splicing events are shown. In vitro cells are primary Cdh5(PAC)-CreERT2; mT/mG cells from the aorta, isolated as described in Materials and methods, cultured in static conditions and sorted by FACs for eGFP+ before RNA isolation. Splicing events in these cells were compared to high-flow carotid artery intimal isolate. In vivo low-flow is the comparison of the low-flow intimal isolate to the high-flow intimal isolate. Pearson correlation indicates the correlation between the change in inclusion observed in low-flow activated endothelial cells in vivo versus low-flow activation of endothelial cells in vitro.
Figure Legend Snippet: Arterial cells cultured in vitro replicate splicing changes observed under low and disturbed flow in vivo. Comparison of the change in inclusion frequency by MISO for individual splicing events of the classes shown (by color code on bottom), relative to high-flow arterial endothelium in vivo. Other splice events include A3SS, A5SS, MXE and RI. Only flow-regulated alternative splicing events are shown. In vitro cells are primary Cdh5(PAC)-CreERT2; mT/mG cells from the aorta, isolated as described in Materials and methods, cultured in static conditions and sorted by FACs for eGFP+ before RNA isolation. Splicing events in these cells were compared to high-flow carotid artery intimal isolate. In vivo low-flow is the comparison of the low-flow intimal isolate to the high-flow intimal isolate. Pearson correlation indicates the correlation between the change in inclusion observed in low-flow activated endothelial cells in vivo versus low-flow activation of endothelial cells in vitro.

Techniques Used: Cell Culture, In Vitro, Flow Cytometry, In Vivo, Isolation, FACS, Activation Assay

28) Product Images from "Disease-relevant transcriptional signatures identified in individual smooth muscle cells from healthy mouse vessels"

Article Title: Disease-relevant transcriptional signatures identified in individual smooth muscle cells from healthy mouse vessels

Journal: Nature Communications

doi: 10.1038/s41467-018-06891-x

Sca1 is upregulated in response to VSMC stimulation in vitro and in vivo. a Relative expression (log 2 -transformed) of Myh11 and Ly6a/Sca1 in ex vivo (black) and cultured mouse aortic VSMCs at passage 4–5 (red) determined by RT-qPCR, normalised to housekeeping gene expression ( Hmbs ). Lines show mean from analysis of three independent primary cultures, error bars show s.e.m. Differences in Myh11 ( p = 0.001) and Ly6a/Sca1 ( p = 5.1e−10) expression are statistically significant (student’s t -test). b The PCA plot of single-cell expression profiles for ex vivo VSMCs (squares) and cultured VSMCs (triangles) shown in Fig. 1e , with expression level of Ly6a/Sca1 colour-coded from light to dark grey. c , d Images ( c ) and GFP-signal quantification ( d ) of FACS-isolated medial cells from Sca1-GFP animals (sorted as GFP-negative, n = 4, top row in c ) with Sca1-GFP adventitial (Adv, sorted as GFP– or GFP+ (middle row in c ), tissue from four animals was pooled) and wildtype (WT) medial cell controls (sorted as GFP-, n = 1, lower row in c ). Cells were cultured for 11 days before fixation and confocal imaging. c Epifluorescence images showing GFP signal after 3 or 10 days of culture. Scale bars are 100 µm. d Quantification of GFP signal in each population showing the number of GFP-positive cells as a percentage of the total number of DAPI-positive cells. Images for quantification were taken in a single z plane. Individual replicates and their mean are indicated and error bars show s.e.m. e Logistic regression analysis of the relationship between S+L+ cells and time after lineage labelling (logit-link logistic regression coefficient = 0.016+/−0.005 [mean+/−95% confidence interval], p -value based on Student’s distribution = 2.56e−10). Trendline and data points, colour-coded by animal age (black gradient), are shown. Age was not included in the regression model presented here; the model accounting for both time after labelling and age is shown in Supplementary Fig. 9 . f , g FACS plots showing EYFP and Sca1 (APC) expression in cells from the left common carotid artery (LCCA) isolated from tamoxifen-labelled Myh11-CreERt2/EYFP no injury controls ( f ) or eight days after ligation ( g ). h The percentage of lineage labelled cells (EYFP+) that expressed Sca1 in the LCCA isolated from ligated and no injury controls. Dots represents data from independent animals ( n = 5 for each group), lines show means, and error bars s.e.m
Figure Legend Snippet: Sca1 is upregulated in response to VSMC stimulation in vitro and in vivo. a Relative expression (log 2 -transformed) of Myh11 and Ly6a/Sca1 in ex vivo (black) and cultured mouse aortic VSMCs at passage 4–5 (red) determined by RT-qPCR, normalised to housekeeping gene expression ( Hmbs ). Lines show mean from analysis of three independent primary cultures, error bars show s.e.m. Differences in Myh11 ( p = 0.001) and Ly6a/Sca1 ( p = 5.1e−10) expression are statistically significant (student’s t -test). b The PCA plot of single-cell expression profiles for ex vivo VSMCs (squares) and cultured VSMCs (triangles) shown in Fig. 1e , with expression level of Ly6a/Sca1 colour-coded from light to dark grey. c , d Images ( c ) and GFP-signal quantification ( d ) of FACS-isolated medial cells from Sca1-GFP animals (sorted as GFP-negative, n = 4, top row in c ) with Sca1-GFP adventitial (Adv, sorted as GFP– or GFP+ (middle row in c ), tissue from four animals was pooled) and wildtype (WT) medial cell controls (sorted as GFP-, n = 1, lower row in c ). Cells were cultured for 11 days before fixation and confocal imaging. c Epifluorescence images showing GFP signal after 3 or 10 days of culture. Scale bars are 100 µm. d Quantification of GFP signal in each population showing the number of GFP-positive cells as a percentage of the total number of DAPI-positive cells. Images for quantification were taken in a single z plane. Individual replicates and their mean are indicated and error bars show s.e.m. e Logistic regression analysis of the relationship between S+L+ cells and time after lineage labelling (logit-link logistic regression coefficient = 0.016+/−0.005 [mean+/−95% confidence interval], p -value based on Student’s distribution = 2.56e−10). Trendline and data points, colour-coded by animal age (black gradient), are shown. Age was not included in the regression model presented here; the model accounting for both time after labelling and age is shown in Supplementary Fig. 9 . f , g FACS plots showing EYFP and Sca1 (APC) expression in cells from the left common carotid artery (LCCA) isolated from tamoxifen-labelled Myh11-CreERt2/EYFP no injury controls ( f ) or eight days after ligation ( g ). h The percentage of lineage labelled cells (EYFP+) that expressed Sca1 in the LCCA isolated from ligated and no injury controls. Dots represents data from independent animals ( n = 5 for each group), lines show means, and error bars s.e.m

Techniques Used: In Vitro, In Vivo, Expressing, Transformation Assay, Ex Vivo, Cell Culture, Quantitative RT-PCR, FACS, Isolation, Imaging, Ligation

VSMC-lineage cells express Sca1. a Schematic showing strategies for lineage labelling of VSMCs using a Myh11-driven, tamoxifen-inducible Cre-recombinase (CreERt2). Tamoxifen treatment activates Cre recombinase activity, resulting in VSMC-specific excision of the stop codon in fluorescent reporter transgenes inserted into the Rosa26 locus (R26). Left panel, the single-colour EYFP reporter. Right panel, the multicolour Confetti reporter, which results in stochastic labelling of VSMC-lineage cells with one of four fluorescent proteins (GFP, YFP, RFP, CFP). b , c Maximum projection of a 12 µm transverse cryosection from the carotid artery of an Myh11-CreERt2/Confetti animal one week after tamoxifen labelling. Confetti fluorescent proteins are shown in red (RFP), yellow (YFP), blue (CFP) and green (nuclear GFP), elastic lamina autofluorescence in green and nuclear DAPI in white. The white boxed region in b is magnified in c , and the dashed lines show the medial-adventitial (red) and medial-endothelial (blue) borders in each panel. Scale bars are 50 µm ( b ) and 10 µm ( c ). d , e FACS plot showing forward scatter (FSC) and EYFP expression in all ( d ) or gated Sca1+ cells ( e ) isolated from the medial layer of aortas from Myh11-CreERt2/Rosa26-EYF P animals
Figure Legend Snippet: VSMC-lineage cells express Sca1. a Schematic showing strategies for lineage labelling of VSMCs using a Myh11-driven, tamoxifen-inducible Cre-recombinase (CreERt2). Tamoxifen treatment activates Cre recombinase activity, resulting in VSMC-specific excision of the stop codon in fluorescent reporter transgenes inserted into the Rosa26 locus (R26). Left panel, the single-colour EYFP reporter. Right panel, the multicolour Confetti reporter, which results in stochastic labelling of VSMC-lineage cells with one of four fluorescent proteins (GFP, YFP, RFP, CFP). b , c Maximum projection of a 12 µm transverse cryosection from the carotid artery of an Myh11-CreERt2/Confetti animal one week after tamoxifen labelling. Confetti fluorescent proteins are shown in red (RFP), yellow (YFP), blue (CFP) and green (nuclear GFP), elastic lamina autofluorescence in green and nuclear DAPI in white. The white boxed region in b is magnified in c , and the dashed lines show the medial-adventitial (red) and medial-endothelial (blue) borders in each panel. Scale bars are 50 µm ( b ) and 10 µm ( c ). d , e FACS plot showing forward scatter (FSC) and EYFP expression in all ( d ) or gated Sca1+ cells ( e ) isolated from the medial layer of aortas from Myh11-CreERt2/Rosa26-EYF P animals

Techniques Used: Activity Assay, FACS, Expressing, Isolation

29) Product Images from "Rap1 and membrane lipids cooperatively recruit talin to trigger integrin activation"

Article Title: Rap1 and membrane lipids cooperatively recruit talin to trigger integrin activation

Journal: Journal of Cell Science

doi: 10.1242/jcs.235531

Rap1 and PIP 2 binding to talin regulate integrin activity in fibroblasts. (A) Schematic overview of ypet-tagged talin variants for re-expression in talin 1/2dKO fibroblasts. (B) Expression levels of talin variants assessed by western blotting. (C) FACS analysis of β1, β3, α5 and αV integrin surface levels and ypet intensity. (D,E) Relative adhesion of talin 1/2dKO fibroblasts expressing ypet alone or ypet-tagged talin variants carrying the indicated mutations on fibronectin (D) and laminin (E) normalized to unspecific adhesion to poly-L-lysine. Values for cells expressing WT talin were set to 1 ( n =8). (F) Spreading area of cells plated on fibronectin for 20, 60 and 120 min ( n =6). (G–I) Quantitative analysis of immunofluorescence images of talin 1/2dKO fibroblasts expressing various talin variants shown as focal adhesion area (G), ypet intensity in paxillin-positive areas relative to total cellular intensity (H) and intensity of anti-β1 integrin 9EG7 antibody signal within adhesion sites (I) ( n =6). Data are presented as mean±95% CI. * P
Figure Legend Snippet: Rap1 and PIP 2 binding to talin regulate integrin activity in fibroblasts. (A) Schematic overview of ypet-tagged talin variants for re-expression in talin 1/2dKO fibroblasts. (B) Expression levels of talin variants assessed by western blotting. (C) FACS analysis of β1, β3, α5 and αV integrin surface levels and ypet intensity. (D,E) Relative adhesion of talin 1/2dKO fibroblasts expressing ypet alone or ypet-tagged talin variants carrying the indicated mutations on fibronectin (D) and laminin (E) normalized to unspecific adhesion to poly-L-lysine. Values for cells expressing WT talin were set to 1 ( n =8). (F) Spreading area of cells plated on fibronectin for 20, 60 and 120 min ( n =6). (G–I) Quantitative analysis of immunofluorescence images of talin 1/2dKO fibroblasts expressing various talin variants shown as focal adhesion area (G), ypet intensity in paxillin-positive areas relative to total cellular intensity (H) and intensity of anti-β1 integrin 9EG7 antibody signal within adhesion sites (I) ( n =6). Data are presented as mean±95% CI. * P

Techniques Used: Binding Assay, Activity Assay, Expressing, Western Blot, FACS, Immunofluorescence

Binding of Rap1 to talin F0 and F1 domains synergize to promote cell adhesion and spreading. (A) Overview of C-terminally EGFP-tagged talin head (TH) constructs. (B) αIIbβ3 integrin activation in CHO A5 cells expressing EGFP alone or EGFP-tagged TH variants assessed by FACS analysis of clone PAC1 antibody binding. Values were normalized to αIIb integrin surface levels. WT values were set to 1. n =5. (C) Schematic overview of ypet-tagged talin variants re-expressed in talin 1/2dKO fibroblasts by retroviral transduction. (D) Western blot analyses of ypet-tagged talin variant-transduced cells for their expression of talin, RIAM and Rap1. (E) FACS analysis of ypet intensity in transduced cell lines. (F) Static adhesion of talin 1/2dKO fibroblasts expressing ypet, ypet-tagged WT talin or variants on fibronectin and laminin. Values of WT talin-transduced cells were set to 1 ( n =12/6). (G) FACS analysis of β1, β3, α5 and αV integrin surface levels in talin variant-transduced cells. (H) Spreading area of talin 1/2dKO fibroblasts expressing ypet, ypet-talin WT and ypet-talin variants ( n =6). (I) Phase contrast images of talin1 fl/fl /talin2 −/− cells and talin 1/2dKO cells expressing talin variants. Scale bar: 25 µm. Data are presented as mean±95% CI. * P
Figure Legend Snippet: Binding of Rap1 to talin F0 and F1 domains synergize to promote cell adhesion and spreading. (A) Overview of C-terminally EGFP-tagged talin head (TH) constructs. (B) αIIbβ3 integrin activation in CHO A5 cells expressing EGFP alone or EGFP-tagged TH variants assessed by FACS analysis of clone PAC1 antibody binding. Values were normalized to αIIb integrin surface levels. WT values were set to 1. n =5. (C) Schematic overview of ypet-tagged talin variants re-expressed in talin 1/2dKO fibroblasts by retroviral transduction. (D) Western blot analyses of ypet-tagged talin variant-transduced cells for their expression of talin, RIAM and Rap1. (E) FACS analysis of ypet intensity in transduced cell lines. (F) Static adhesion of talin 1/2dKO fibroblasts expressing ypet, ypet-tagged WT talin or variants on fibronectin and laminin. Values of WT talin-transduced cells were set to 1 ( n =12/6). (G) FACS analysis of β1, β3, α5 and αV integrin surface levels in talin variant-transduced cells. (H) Spreading area of talin 1/2dKO fibroblasts expressing ypet, ypet-talin WT and ypet-talin variants ( n =6). (I) Phase contrast images of talin1 fl/fl /talin2 −/− cells and talin 1/2dKO cells expressing talin variants. Scale bar: 25 µm. Data are presented as mean±95% CI. * P

Techniques Used: Binding Assay, Construct, Activation Assay, Expressing, FACS, Transduction, Western Blot, Variant Assay

30) Product Images from "Smad3 Differentially Regulates the Induction of Regulatory and Inflammatory T Cell Differentiation *"

Article Title: Smad3 Differentially Regulates the Induction of Regulatory and Inflammatory T Cell Differentiation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.C109.078238

Smad3 deficiency enhances Th17 cell differentiation in the presence of high doses of TGF-β. A–C , FACS-sorted naive CD4 + CD25 − CD62L hi CD44 lo T cells from Smad3 WT or KO mice were activated under indicated Th17 conditions for 4 days.
Figure Legend Snippet: Smad3 deficiency enhances Th17 cell differentiation in the presence of high doses of TGF-β. A–C , FACS-sorted naive CD4 + CD25 − CD62L hi CD44 lo T cells from Smad3 WT or KO mice were activated under indicated Th17 conditions for 4 days.

Techniques Used: Cell Differentiation, FACS, Mouse Assay

31) Product Images from "Interleukin 21 collaborates with interferon-γ for the optimal expression of interferon-stimulated genes and enhances protection against enteric microbial infection"

Article Title: Interleukin 21 collaborates with interferon-γ for the optimal expression of interferon-stimulated genes and enhances protection against enteric microbial infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007614

The requirement of IFN-γ, but not IFN-α/β, for the clearance of infection with C . rodentium . A . The expression levels of interferons (α, β, γ, λ) by the FACS-sorted CD4 + T cells (left) and DCs (right) isolated from the distal colonic LP of WT mice 9 days p.i. Results are from pooled distal colon LP of WT mice ( n = 25) 9 days after infection with C . rodentium analyzed by Nanostring. N.D., not detected. B . Cytokine ELISA kinetics of IFN-α, IFN-γ and IL-17A from supernatants of the ex-vivo organ culture of the colonic tissues from WT or Il21r -/- mice collected at 9 days p.i. (sampled at times indicated during the 180 hr cultures). Data are the Mean ± SEM of one experiment from two independent experiments with a total of 10 ( Il21r -/- ) or 10 (WT) mice/group. C . Bacterial burden in the feces of C . rodentium -infected Ifnar -/- versus WT controls as shown by colony forming units (CFU)/g feces. D . Bacterial infection kinetics in the feces and E . changes in body weight of Ifng -/- mice versus WT controls following infection with C . rodentium . The dashed line represents the sensitivity of the culture method. The results are the Mean ± SEM of 5 mice per group. * p
Figure Legend Snippet: The requirement of IFN-γ, but not IFN-α/β, for the clearance of infection with C . rodentium . A . The expression levels of interferons (α, β, γ, λ) by the FACS-sorted CD4 + T cells (left) and DCs (right) isolated from the distal colonic LP of WT mice 9 days p.i. Results are from pooled distal colon LP of WT mice ( n = 25) 9 days after infection with C . rodentium analyzed by Nanostring. N.D., not detected. B . Cytokine ELISA kinetics of IFN-α, IFN-γ and IL-17A from supernatants of the ex-vivo organ culture of the colonic tissues from WT or Il21r -/- mice collected at 9 days p.i. (sampled at times indicated during the 180 hr cultures). Data are the Mean ± SEM of one experiment from two independent experiments with a total of 10 ( Il21r -/- ) or 10 (WT) mice/group. C . Bacterial burden in the feces of C . rodentium -infected Ifnar -/- versus WT controls as shown by colony forming units (CFU)/g feces. D . Bacterial infection kinetics in the feces and E . changes in body weight of Ifng -/- mice versus WT controls following infection with C . rodentium . The dashed line represents the sensitivity of the culture method. The results are the Mean ± SEM of 5 mice per group. * p

Techniques Used: Infection, Expressing, FACS, Isolation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Ex Vivo, Organ Culture

32) Product Images from "Massive clonal expansion of medulloblastoma-specific T cells during adoptive cellular therapy"

Article Title: Massive clonal expansion of medulloblastoma-specific T cells during adoptive cellular therapy

Journal: Science Advances

doi: 10.1126/sciadv.aav9879

Selective expansion of the tumor-reactive TCR Vβ family in mice responsive to ACT. To generate antitumor T cells, total RNA is extracted from tumor cells and electroporated into syngeneic bone marrow–derived DCs. These cells are then cocultured with splenocytes from a previously immunized mouse with interleukin-2 for 5 to 7 days generating a polyclonal population of CD8 + T cells. After this ex vivo activation, 10 7 T cells are adoptively transferred into tumor-bearing mice followed by vaccination with 2.5 × 10 5 RNA-pulsed DCs. In the preclinical model of ACT, C57BL/6 mice receive orthotopic tumor followed by host conditioning with total body irradiation and hematopoietic stem cell transfer to protect from bone marrow failure. ( A ) Mice implanted with cerebellar NSC medulloblastoma were treated with ACT using DsRed + tumor–reactive T cells. Spleens were harvested from all mice, and relative abundance of each TCR Vβ family was measured in both responders and nonresponders. Here, 25 mice are implanted with tumor and treated with ACT. The first five nonresponders that succumb to tumor are taken at humane end point and spleens were analyzed. The five responders are treated mice that demonstrate no evidence of tumor after 120 days. This experiment was repeated twice with the same results as shown. n = 5 to 7 mice per group. ( B ) Spleens of five asymptomatic long-term survivors were harvested at 90 days after ACT. DsRed + T cells were isolated and separated by the TCR Vβ family. Each TCR Vβ family was cocultured in vitro against tumor cells, and IFN-γ secretion was measured. ( C ) Splenic T lymphocytes were harvested from nonresponders to therapy upon detection of tumor via bioluminescent imaging. DsRed + T cells were FACS-isolated and sorted into TCR Vβ families and then used as effectors against the primary NSC cell line. IFN-γ was measured to determine antitumor reactivity.
Figure Legend Snippet: Selective expansion of the tumor-reactive TCR Vβ family in mice responsive to ACT. To generate antitumor T cells, total RNA is extracted from tumor cells and electroporated into syngeneic bone marrow–derived DCs. These cells are then cocultured with splenocytes from a previously immunized mouse with interleukin-2 for 5 to 7 days generating a polyclonal population of CD8 + T cells. After this ex vivo activation, 10 7 T cells are adoptively transferred into tumor-bearing mice followed by vaccination with 2.5 × 10 5 RNA-pulsed DCs. In the preclinical model of ACT, C57BL/6 mice receive orthotopic tumor followed by host conditioning with total body irradiation and hematopoietic stem cell transfer to protect from bone marrow failure. ( A ) Mice implanted with cerebellar NSC medulloblastoma were treated with ACT using DsRed + tumor–reactive T cells. Spleens were harvested from all mice, and relative abundance of each TCR Vβ family was measured in both responders and nonresponders. Here, 25 mice are implanted with tumor and treated with ACT. The first five nonresponders that succumb to tumor are taken at humane end point and spleens were analyzed. The five responders are treated mice that demonstrate no evidence of tumor after 120 days. This experiment was repeated twice with the same results as shown. n = 5 to 7 mice per group. ( B ) Spleens of five asymptomatic long-term survivors were harvested at 90 days after ACT. DsRed + T cells were isolated and separated by the TCR Vβ family. Each TCR Vβ family was cocultured in vitro against tumor cells, and IFN-γ secretion was measured. ( C ) Splenic T lymphocytes were harvested from nonresponders to therapy upon detection of tumor via bioluminescent imaging. DsRed + T cells were FACS-isolated and sorted into TCR Vβ families and then used as effectors against the primary NSC cell line. IFN-γ was measured to determine antitumor reactivity.

Techniques Used: Mouse Assay, Activated Clotting Time Assay, Derivative Assay, Ex Vivo, Activation Assay, Irradiation, Isolation, In Vitro, Imaging, FACS

Identification of tumor-specific T lymphocytes in a preclinical glioma model. ( A ) Tumor-reactive T cells were generated in vitro and separated into 15 TCR Vβ families using sterile FACS isolation. T cells (4 × 10 5 ) per Vβ family were cocultured against 4 × 10 4 KR158B tumor target cells overnight, and supernatant IFN-γ was measured as an indication of the recognition of cognate tumor antigen. All conditions were conducted in triplicate, and the experiment was repeated an additional three times with the same results. ( B ) Fifteen mice received ACT using DsRed + tumor–reactive T cells. Relative frequencies of TCR Vβ families within the adoptively transferred DsRed + T cell population were compared between the first five nonresponders to therapy and five long-term survivors with no signs of tumor. ( C ) Spleens of the asymptomatic long-term survivors were also harvested for DsRed + T cells, which were further separated by the TCR Vβ family using FACS. Each TCR Vβ family was cocultured in vitro against tumor cells as above, and IFN-γ secretion was measured.
Figure Legend Snippet: Identification of tumor-specific T lymphocytes in a preclinical glioma model. ( A ) Tumor-reactive T cells were generated in vitro and separated into 15 TCR Vβ families using sterile FACS isolation. T cells (4 × 10 5 ) per Vβ family were cocultured against 4 × 10 4 KR158B tumor target cells overnight, and supernatant IFN-γ was measured as an indication of the recognition of cognate tumor antigen. All conditions were conducted in triplicate, and the experiment was repeated an additional three times with the same results. ( B ) Fifteen mice received ACT using DsRed + tumor–reactive T cells. Relative frequencies of TCR Vβ families within the adoptively transferred DsRed + T cell population were compared between the first five nonresponders to therapy and five long-term survivors with no signs of tumor. ( C ) Spleens of the asymptomatic long-term survivors were also harvested for DsRed + T cells, which were further separated by the TCR Vβ family using FACS. Each TCR Vβ family was cocultured in vitro against tumor cells as above, and IFN-γ secretion was measured.

Techniques Used: Generated, In Vitro, FACS, Isolation, Mouse Assay, Activated Clotting Time Assay

33) Product Images from "Anti-apoptotic ARC protein confers chemoresistance by controlling leukemia-microenvironment interactions through a NFκB/IL1β signaling network"

Article Title: Anti-apoptotic ARC protein confers chemoresistance by controlling leukemia-microenvironment interactions through a NFκB/IL1β signaling network

Journal: Oncotarget

doi: 10.18632/oncotarget.7911

MSCs induce the expression of IL1β in AML cells OCI-AML3 cells were cultured alone or co-cultured with ARC KD or control MSCs for 48 h. CD45 + CD90 − AML cells were FACS-sorted from the floaters (collected from cells in the suspension and after PBS wash) and attached (collected by trypsinization) cells. IL1β RNA levels were determined in sorted OCI-AML3 cells and the cells cultured alone by real-time RT-PCR.
Figure Legend Snippet: MSCs induce the expression of IL1β in AML cells OCI-AML3 cells were cultured alone or co-cultured with ARC KD or control MSCs for 48 h. CD45 + CD90 − AML cells were FACS-sorted from the floaters (collected from cells in the suspension and after PBS wash) and attached (collected by trypsinization) cells. IL1β RNA levels were determined in sorted OCI-AML3 cells and the cells cultured alone by real-time RT-PCR.

Techniques Used: Expressing, Cell Culture, FACS, Quantitative RT-PCR

ARC in AML modulates CCL2, CCL4, and CXCL12 expression in MSCs ( A ) MSCs were cultured alone or with ARC KD OCI-AML3, ARC OE KG-1, or the respective control cells for 48 h and MSCs were FACS sorted conservatively as marked in the boxes for CD45 − CD90 + cells. ( B ) CCL2, CCL4, and CXCL12 levels in MSCs were determined by quantitative RT-PCR.
Figure Legend Snippet: ARC in AML modulates CCL2, CCL4, and CXCL12 expression in MSCs ( A ) MSCs were cultured alone or with ARC KD OCI-AML3, ARC OE KG-1, or the respective control cells for 48 h and MSCs were FACS sorted conservatively as marked in the boxes for CD45 − CD90 + cells. ( B ) CCL2, CCL4, and CXCL12 levels in MSCs were determined by quantitative RT-PCR.

Techniques Used: Expressing, Cell Culture, FACS, Quantitative RT-PCR

34) Product Images from "RORγt+ cells selectively express redundant cation channels linked to the Golgi apparatus"

Article Title: RORγt+ cells selectively express redundant cation channels linked to the Golgi apparatus

Journal: Scientific Reports

doi: 10.1038/srep23682

Tmem176b single-deficient mouse susceptibility to the development of EAE, chronic and atute colitis. ( a ) EAE was induced in WT (n = 6) and Tmem176b −/− (n = 8) mice by immunisation (s.c.) with MOG peptide in CFA. Clinical course of disease is shown. ( b ) Chronic colitis was induced in Rag1 −/− mice (n = 11–12 in each group) by adoptive transfer (i.v.) of FACS-sorted CD4 + CD45RB hi T cells from WT or Tmem176b −/− mice. Data are presented as percent of initial weight. ( c ) Acute colitis was induced in WT (n = 6) and Tmem176b −/− (n = 8) mice with 3% DSS in drinking water for 5 consecutive days. Data are presented as percent of initial weight.
Figure Legend Snippet: Tmem176b single-deficient mouse susceptibility to the development of EAE, chronic and atute colitis. ( a ) EAE was induced in WT (n = 6) and Tmem176b −/− (n = 8) mice by immunisation (s.c.) with MOG peptide in CFA. Clinical course of disease is shown. ( b ) Chronic colitis was induced in Rag1 −/− mice (n = 11–12 in each group) by adoptive transfer (i.v.) of FACS-sorted CD4 + CD45RB hi T cells from WT or Tmem176b −/− mice. Data are presented as percent of initial weight. ( c ) Acute colitis was induced in WT (n = 6) and Tmem176b −/− (n = 8) mice with 3% DSS in drinking water for 5 consecutive days. Data are presented as percent of initial weight.

Techniques Used: Mouse Assay, Adoptive Transfer Assay, FACS

Tmem176b single-deficient mouse susceptibility to the development of psoriasis-like dermatitis. ( a ) Psoriasis-like dermatitis was induced in WT mice by topical application of imiquimod (IMQ) cream on the shaved back skin. At day 4, draining (inguinal) lymph nodes were harvested and TCRγδ + and TCRβ + CD4 + T cells were FACS-sorted. ( b ) Expression of indicated genes was assessed by quantitative RT-PCR. Each dot represents an individual mouse (n = 6 in each group). Statistically significant differences are indicated: **p
Figure Legend Snippet: Tmem176b single-deficient mouse susceptibility to the development of psoriasis-like dermatitis. ( a ) Psoriasis-like dermatitis was induced in WT mice by topical application of imiquimod (IMQ) cream on the shaved back skin. At day 4, draining (inguinal) lymph nodes were harvested and TCRγδ + and TCRβ + CD4 + T cells were FACS-sorted. ( b ) Expression of indicated genes was assessed by quantitative RT-PCR. Each dot represents an individual mouse (n = 6 in each group). Statistically significant differences are indicated: **p

Techniques Used: Mouse Assay, FACS, Expressing, Quantitative RT-PCR

Tmem176a and b mRNA expression in mouse and human T cells. ( a ) Conventional GFP − (Foxp3 − Tconv) or regulatory GFP + (Foxp3 + Treg) CD4 + T cells were FACS-sorted from the spleen or intestinal lamina propria (small intestine and colon) of Foxp3 EGFP mice. As expected, the population of Nrp1 −/low “adaptive” peripherally Tregs is dominant in the intestines. Conversely, Nrp1 + “natural” thymically derived Tregs represent the major population of Tregs in spleen. ( b ) Expression of indicated genes was assessed by quantitative RT-PCR. Each dot represents an individual mouse (n = 6–7 in each group). Statistically significant differences between intestinal Tconv and Treg are indicated: *p
Figure Legend Snippet: Tmem176a and b mRNA expression in mouse and human T cells. ( a ) Conventional GFP − (Foxp3 − Tconv) or regulatory GFP + (Foxp3 + Treg) CD4 + T cells were FACS-sorted from the spleen or intestinal lamina propria (small intestine and colon) of Foxp3 EGFP mice. As expected, the population of Nrp1 −/low “adaptive” peripherally Tregs is dominant in the intestines. Conversely, Nrp1 + “natural” thymically derived Tregs represent the major population of Tregs in spleen. ( b ) Expression of indicated genes was assessed by quantitative RT-PCR. Each dot represents an individual mouse (n = 6–7 in each group). Statistically significant differences between intestinal Tconv and Treg are indicated: *p

Techniques Used: Expressing, FACS, Mouse Assay, Derivative Assay, Quantitative RT-PCR

35) Product Images from "Genetic Interaction Between Site-Specific Epigenetic Marks and Roles of H4v in Transcription Termination in Trypanosoma brucei"

Article Title: Genetic Interaction Between Site-Specific Epigenetic Marks and Roles of H4v in Transcription Termination in Trypanosoma brucei

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2021.744878

Global DNA replication impairment in TKO cells. Replication was examined by marker frequency analysis followed by high-throughput sequencing (MFA-seq). TKO strain with a floxed H3v-HA allele was treated with tetracycline for 0, 1, or 2 days and stained with PI. Cells in G1, early S, late S, and G2 phase were FACS-sorted ( Supplementary Figure 8C ). Genomic DNA was prepared and sequenced in the Illumina platform. Sequence reads were aligned to the Lister 427 genome and analyzed with sliding window (10-kb bin, 2.5-kb step). Read count ratio between early S to G1 was plotted for the whole genome, including subtelomeric regions. Chromosome cores (pink) and subtelomeres (gray) in the chromosome diagram are shown. Several of early replicating origins occur in subtelomeres of chromosomes 10, 11, and 9 (red arrows in the WT plot). MFA-seq reads obtained from WT ( Kim, 2019 ) were re-analyzed and mapped to the Lister 427 genome for comparison. Subtelomere 6A of chromosome 6 has been lost in the TKO strain.
Figure Legend Snippet: Global DNA replication impairment in TKO cells. Replication was examined by marker frequency analysis followed by high-throughput sequencing (MFA-seq). TKO strain with a floxed H3v-HA allele was treated with tetracycline for 0, 1, or 2 days and stained with PI. Cells in G1, early S, late S, and G2 phase were FACS-sorted ( Supplementary Figure 8C ). Genomic DNA was prepared and sequenced in the Illumina platform. Sequence reads were aligned to the Lister 427 genome and analyzed with sliding window (10-kb bin, 2.5-kb step). Read count ratio between early S to G1 was plotted for the whole genome, including subtelomeric regions. Chromosome cores (pink) and subtelomeres (gray) in the chromosome diagram are shown. Several of early replicating origins occur in subtelomeres of chromosomes 10, 11, and 9 (red arrows in the WT plot). MFA-seq reads obtained from WT ( Kim, 2019 ) were re-analyzed and mapped to the Lister 427 genome for comparison. Subtelomere 6A of chromosome 6 has been lost in the TKO strain.

Techniques Used: Marker, Next-Generation Sequencing, Staining, FACS, Sequencing

36) Product Images from "Strong Expansion of Human Regulatory T Cells for Adoptive Cell Therapy Results in Epigenetic Changes Which May Impact Their Survival and Function"

Article Title: Strong Expansion of Human Regulatory T Cells for Adoptive Cell Therapy Results in Epigenetic Changes Which May Impact Their Survival and Function

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2021.751590

(A) Change in mean DMR methylation of shared DMRs over the 23 days of in vitro expansion. Early DMRs were defined as being significantly differentially methylated in day 10 – day 0 (and remained significant in day 23 – day 0), and Late DMRs were only significantly differentially methylated at day 23 – day 0. Each line displays one DMR, bold black line displays the mean. (B) Display of all shared DMRs according to their directionality of methylation change (hyper/hypo) and to their Early/Late classification (as in A ) in production Run1 and 2. Color code indicates genic annotation of the DMR. (C) Unsupervised hierarchical clustering of shared DMRs in both production runs, with DMRs mapping to genes implicated in T cell activation and T cell differentiation (GO terms 0042110 and 0030217) highlighted. (D) Gene ontology enrichment analysis of shared DMRs that were consistently “Early” or “Late” between both production runs, stratified by directionality. The top 5 GO terms (ranked by adjusted p -value) for each DMR class are represented, many of which overlap. Size of dots indicates number of genes associated with GO term, and color indicates adjusted p -value. (E–G) Level of DNA methylation on selected DMRs in known T cell genes. (H) DNA methylation at the promoters of RHOH , HLX , and RARA in FACS-sorted Tregs and memory CD4 + T cells (CD4mem) before (Day 0) and after frequent CD3/CD28 stimulation (Day 23). ∗ denotes p .val
Figure Legend Snippet: (A) Change in mean DMR methylation of shared DMRs over the 23 days of in vitro expansion. Early DMRs were defined as being significantly differentially methylated in day 10 – day 0 (and remained significant in day 23 – day 0), and Late DMRs were only significantly differentially methylated at day 23 – day 0. Each line displays one DMR, bold black line displays the mean. (B) Display of all shared DMRs according to their directionality of methylation change (hyper/hypo) and to their Early/Late classification (as in A ) in production Run1 and 2. Color code indicates genic annotation of the DMR. (C) Unsupervised hierarchical clustering of shared DMRs in both production runs, with DMRs mapping to genes implicated in T cell activation and T cell differentiation (GO terms 0042110 and 0030217) highlighted. (D) Gene ontology enrichment analysis of shared DMRs that were consistently “Early” or “Late” between both production runs, stratified by directionality. The top 5 GO terms (ranked by adjusted p -value) for each DMR class are represented, many of which overlap. Size of dots indicates number of genes associated with GO term, and color indicates adjusted p -value. (E–G) Level of DNA methylation on selected DMRs in known T cell genes. (H) DNA methylation at the promoters of RHOH , HLX , and RARA in FACS-sorted Tregs and memory CD4 + T cells (CD4mem) before (Day 0) and after frequent CD3/CD28 stimulation (Day 23). ∗ denotes p .val

Techniques Used: Methylation, In Vitro, Activation Assay, Cell Differentiation, DNA Methylation Assay, FACS

(A) FACS-sorted Tregs and CD4 memory T cells (Day 0) were analyzed for CpG methylation at Treg specific demethylated regions (Treg-DRs). Significantly differentially methylated CpGs ( p . val
Figure Legend Snippet: (A) FACS-sorted Tregs and CD4 memory T cells (Day 0) were analyzed for CpG methylation at Treg specific demethylated regions (Treg-DRs). Significantly differentially methylated CpGs ( p . val

Techniques Used: FACS, CpG Methylation Assay, Methylation

37) Product Images from "Site-specific epigenetic marks in Trypanosoma brucei transcription termination, antigenic variation, and proliferation"

Article Title: Site-specific epigenetic marks in Trypanosoma brucei transcription termination, antigenic variation, and proliferation

Journal: bioRxiv

doi: 10.1101/2021.05.13.444086

Global DNA replication impairment in TKO cells. Replication was examined by MFA-seq. TKO strain with floxed H3v-HA allele was treated with tetracycline for 0, 1 or 2 days and stained with PI. Cells in G1, early S, late S and G2 phase were FACS-sorted (Supplementary Figure 9). Genomic DNA was prepared and sequenced in the Illumina platform. Sequence reads were aligned to the Lister 427 genome and analyzed with sliding window (10kb bin, 2.5kb step). Read count ratio between early S to G1 was plotted for the whole genome, including subtelomeric regions. Shown: chromosome cores (pink) and subtelomeres (grey) in the chromosome diagram. Several of early replicating origins occur in subtelomeres of chromosome 10, 11, and 9 (red arrows). MFA-seq reads obtained from WT 32 was re-analyzed and mapped to the Lister 427 genome for comparison. Subtelomere 6A of chromosome 6 is lost in the TKO strain.
Figure Legend Snippet: Global DNA replication impairment in TKO cells. Replication was examined by MFA-seq. TKO strain with floxed H3v-HA allele was treated with tetracycline for 0, 1 or 2 days and stained with PI. Cells in G1, early S, late S and G2 phase were FACS-sorted (Supplementary Figure 9). Genomic DNA was prepared and sequenced in the Illumina platform. Sequence reads were aligned to the Lister 427 genome and analyzed with sliding window (10kb bin, 2.5kb step). Read count ratio between early S to G1 was plotted for the whole genome, including subtelomeric regions. Shown: chromosome cores (pink) and subtelomeres (grey) in the chromosome diagram. Several of early replicating origins occur in subtelomeres of chromosome 10, 11, and 9 (red arrows). MFA-seq reads obtained from WT 32 was re-analyzed and mapped to the Lister 427 genome for comparison. Subtelomere 6A of chromosome 6 is lost in the TKO strain.

Techniques Used: Staining, FACS, Sequencing

38) Product Images from "Anti-Leukemic Properties of Histamine in Monocytic Leukemia: The Role of NOX2"

Article Title: Anti-Leukemic Properties of Histamine in Monocytic Leukemia: The Role of NOX2

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2018.00218

Histamine dihydrochloride (HDC)-induced differentiation of leukemic cells is NOX2-dependent. (A) FACS-plots showing NOX2 and H 2 R expression on wild-type (WT) and NOX2 -KO PLB-985 cells. Expression of CD11b (B,C) , FPR1 (D) , and FPR2 (E) on WT and NOX2 -KO PLB-985 cells cultured in the presence or absence of HDC or dimethyl sulfoxide (DMSO) as determined by flow cytometry. (F) FACS-plot showing NOX2 and H 2 R expression by OCI-AML3 cells. Expression of CD11b (G) , CD14 (H) , FPR1 (I) , and FPR2 (J) on OCI-AML3 cells cultured in the presence or absence of HDC or DMSO. Abbreviations: MFI, median fluorescence intensity. ANOVA; * p
Figure Legend Snippet: Histamine dihydrochloride (HDC)-induced differentiation of leukemic cells is NOX2-dependent. (A) FACS-plots showing NOX2 and H 2 R expression on wild-type (WT) and NOX2 -KO PLB-985 cells. Expression of CD11b (B,C) , FPR1 (D) , and FPR2 (E) on WT and NOX2 -KO PLB-985 cells cultured in the presence or absence of HDC or dimethyl sulfoxide (DMSO) as determined by flow cytometry. (F) FACS-plot showing NOX2 and H 2 R expression by OCI-AML3 cells. Expression of CD11b (G) , CD14 (H) , FPR1 (I) , and FPR2 (J) on OCI-AML3 cells cultured in the presence or absence of HDC or DMSO. Abbreviations: MFI, median fluorescence intensity. ANOVA; * p

Techniques Used: FACS, Expressing, Cell Culture, Flow Cytometry, Cytometry, Fluorescence

Histamine dihydrochloride (HDC) facilitates the differentiation of monocytic primary leukemic cells and may be preferentially efficacious in monocytic forms of leukemia. FACS-plots showing live peripheral blood mononuclear cells from representative newly diagnosed patients with (A) FAB-M0 acute myeloid leukemia (AML) with a dominant immature leukemic population (CD34 + CD33 − CD14 − ) and (B) FAB-M4 AML with two distinct populations: an immature blast population (CD34 + CD33 − CD14 − ) and a mature monocytic population (CD34 − CD33 + CD14 + ). The expression of (C) H 2 R, (D) NOX2, (E) FPR1, and (F) FPR2 on primary AML cells [gated as indicated in (A,B) ] and monocytes from healthy donors was determined by flow cytometry. The M5 leukemia is represented by an open circle. One-way ANOVA. (G–I) Median fluorescence intensity as determined by flow cytometry of (G) HLA-DR, (H) FPR1, and (I) FPR2 on live primary monocytic AML cells (FAB: M4/M5) or non-monocytic AML cells (FAB: M0–M2) cultured for 5 days with GM-CSF/IL-4 in the presence or absence of HDC. Wilcoxon matched pair’s test. * p
Figure Legend Snippet: Histamine dihydrochloride (HDC) facilitates the differentiation of monocytic primary leukemic cells and may be preferentially efficacious in monocytic forms of leukemia. FACS-plots showing live peripheral blood mononuclear cells from representative newly diagnosed patients with (A) FAB-M0 acute myeloid leukemia (AML) with a dominant immature leukemic population (CD34 + CD33 − CD14 − ) and (B) FAB-M4 AML with two distinct populations: an immature blast population (CD34 + CD33 − CD14 − ) and a mature monocytic population (CD34 − CD33 + CD14 + ). The expression of (C) H 2 R, (D) NOX2, (E) FPR1, and (F) FPR2 on primary AML cells [gated as indicated in (A,B) ] and monocytes from healthy donors was determined by flow cytometry. The M5 leukemia is represented by an open circle. One-way ANOVA. (G–I) Median fluorescence intensity as determined by flow cytometry of (G) HLA-DR, (H) FPR1, and (I) FPR2 on live primary monocytic AML cells (FAB: M4/M5) or non-monocytic AML cells (FAB: M0–M2) cultured for 5 days with GM-CSF/IL-4 in the presence or absence of HDC. Wilcoxon matched pair’s test. * p

Techniques Used: FACS, Expressing, Flow Cytometry, Cytometry, Fluorescence, Cell Culture

39) Product Images from "Parallel clonal and molecular profiling of hematopoietic stem cells using RNA barcoding"

Article Title: Parallel clonal and molecular profiling of hematopoietic stem cells using RNA barcoding

Journal: bioRxiv

doi: 10.1101/2022.05.16.491933

A. Violin plots showing the distribution of read count per cell, number of genes per single cell and fraction of mitochondrial genes in single cells within 3 analysed BM populations B. UMAP plot representing computationally assigned clusters of cells based on their transcriptomes (Seurat clusters) C. UMAP plot representing 3 clusters of cells split by their FACS phenotype D. UMAP plots representing the expression of marker genes for HSC (Procr, Slamf1, CD48, Mpl, Selp), MkP (Mpl, Slamf1, Selp) and CFU-E (Hba-a1, Slc25a21)
Figure Legend Snippet: A. Violin plots showing the distribution of read count per cell, number of genes per single cell and fraction of mitochondrial genes in single cells within 3 analysed BM populations B. UMAP plot representing computationally assigned clusters of cells based on their transcriptomes (Seurat clusters) C. UMAP plot representing 3 clusters of cells split by their FACS phenotype D. UMAP plots representing the expression of marker genes for HSC (Procr, Slamf1, CD48, Mpl, Selp), MkP (Mpl, Slamf1, Selp) and CFU-E (Hba-a1, Slc25a21)

Techniques Used: FACS, Expressing, Marker

A. Gating strategy for FACS-purification of single BM populations: HSC, CFU-E and MkP, which were sorted as GFP + . B.-E. Venn diagrams representing the overlap between dominant barcodes (top 90% barcodes identified in all PB samples at 12 and 28-weeks post transplantation) detected in anucleate cells (platelets or erythroid cells) and their BM progenitors (MkP or CFU-E) in mouse 1-4
Figure Legend Snippet: A. Gating strategy for FACS-purification of single BM populations: HSC, CFU-E and MkP, which were sorted as GFP + . B.-E. Venn diagrams representing the overlap between dominant barcodes (top 90% barcodes identified in all PB samples at 12 and 28-weeks post transplantation) detected in anucleate cells (platelets or erythroid cells) and their BM progenitors (MkP or CFU-E) in mouse 1-4

Techniques Used: FACS, Purification, Transplantation Assay

Clonal tracking in blood and bone marrow cells exerts high overlap between detected clones A. UMAP plot showing single cell transcriptomes of sorted BM populations in which barcodes (BC) were detected (all cells which had over 50 000 reads and mitochondrial gene count
Figure Legend Snippet: Clonal tracking in blood and bone marrow cells exerts high overlap between detected clones A. UMAP plot showing single cell transcriptomes of sorted BM populations in which barcodes (BC) were detected (all cells which had over 50 000 reads and mitochondrial gene count

Techniques Used: Clone Assay

A. Gating strategy for PB cell population FACS-purification. Displayed are gates within the MNC/singlet/viable cell population. FACS definitions for purified populations: Mac1 + (CD19 - CD4/8 - CD150 - CD41 - CD45.1 + ), CD19 + (Mac1 - CD4/8 - CD19 + CD150 - CD41 - CD45.1 + ) B. Purity of sorted Mac1 + cells evaluated by FACS Purity of CD19 + cells evaluated by FACS
Figure Legend Snippet: A. Gating strategy for PB cell population FACS-purification. Displayed are gates within the MNC/singlet/viable cell population. FACS definitions for purified populations: Mac1 + (CD19 - CD4/8 - CD150 - CD41 - CD45.1 + ), CD19 + (Mac1 - CD4/8 - CD19 + CD150 - CD41 - CD45.1 + ) B. Purity of sorted Mac1 + cells evaluated by FACS Purity of CD19 + cells evaluated by FACS

Techniques Used: FACS, Purification

In vitro and in vivo validation of RNA barcoding approach for stable, long-term clonal kinetics studies of hematopoiesis A.Experimental set up for in vitro validation of quantitative RNA based clonal studies. The K562 cell line was used for the transduction, single eGFP+ cells were FACS-sorted and expanded to generate monoclonal calibration samples. Four clones were used to create samples, where cells carrying different barcodes were mixed in known ratios-spiked in clone contributing 0.1%, 1%, 5%, 10%, 20% or 50% of total mix. B. Generalised linear model (GLM) correlation between the read count for barcoded transcript retrieved in calibration samples from cDNA or gDNA. C. Experimental setup to test the silencing of eGFP in VavCre x Rosa26tdTomato LSK cells D.-H. eGFP+ chimerism changes in tdTomato+ anucleate cells and CD45.2+ (donor derived) nucleated cells. To calculate the silencing in specific lineage we subtracted eGFP + cells from CD45.2 + cells (nucleated cells-CD19 + , Mac1 + or CD4/8 + cells) or from tdTomato + cells (all donor derived anucleate cells), shown are results from a single experiment in 3 recipient mice
Figure Legend Snippet: In vitro and in vivo validation of RNA barcoding approach for stable, long-term clonal kinetics studies of hematopoiesis A.Experimental set up for in vitro validation of quantitative RNA based clonal studies. The K562 cell line was used for the transduction, single eGFP+ cells were FACS-sorted and expanded to generate monoclonal calibration samples. Four clones were used to create samples, where cells carrying different barcodes were mixed in known ratios-spiked in clone contributing 0.1%, 1%, 5%, 10%, 20% or 50% of total mix. B. Generalised linear model (GLM) correlation between the read count for barcoded transcript retrieved in calibration samples from cDNA or gDNA. C. Experimental setup to test the silencing of eGFP in VavCre x Rosa26tdTomato LSK cells D.-H. eGFP+ chimerism changes in tdTomato+ anucleate cells and CD45.2+ (donor derived) nucleated cells. To calculate the silencing in specific lineage we subtracted eGFP + cells from CD45.2 + cells (nucleated cells-CD19 + , Mac1 + or CD4/8 + cells) or from tdTomato + cells (all donor derived anucleate cells), shown are results from a single experiment in 3 recipient mice

Techniques Used: In Vitro, In Vivo, Transduction, FACS, Clone Assay, Derivative Assay, Mouse Assay

40) Product Images from "Parallel clonal and molecular profiling of hematopoietic stem cells using RNA barcoding"

Article Title: Parallel clonal and molecular profiling of hematopoietic stem cells using RNA barcoding

Journal: bioRxiv

doi: 10.1101/2022.05.16.491933

A. Violin plots showing the distribution of read count per cell, number of genes per single cell and fraction of mitochondrial genes in single cells within 3 analysed BM populations B. UMAP plot representing computationally assigned clusters of cells based on their transcriptomes (Seurat clusters) C. UMAP plot representing 3 clusters of cells split by their FACS phenotype D. UMAP plots representing the expression of marker genes for HSC (Procr, Slamf1, CD48, Mpl, Selp), MkP (Mpl, Slamf1, Selp) and CFU-E (Hba-a1, Slc25a21)
Figure Legend Snippet: A. Violin plots showing the distribution of read count per cell, number of genes per single cell and fraction of mitochondrial genes in single cells within 3 analysed BM populations B. UMAP plot representing computationally assigned clusters of cells based on their transcriptomes (Seurat clusters) C. UMAP plot representing 3 clusters of cells split by their FACS phenotype D. UMAP plots representing the expression of marker genes for HSC (Procr, Slamf1, CD48, Mpl, Selp), MkP (Mpl, Slamf1, Selp) and CFU-E (Hba-a1, Slc25a21)

Techniques Used: FACS, Expressing, Marker

A. Gating strategy for FACS-purification of single BM populations: HSC, CFU-E and MkP, which were sorted as GFP + . B.-E. Venn diagrams representing the overlap between dominant barcodes (top 90% barcodes identified in all PB samples at 12 and 28-weeks post transplantation) detected in anucleate cells (platelets or erythroid cells) and their BM progenitors (MkP or CFU-E) in mouse 1-4
Figure Legend Snippet: A. Gating strategy for FACS-purification of single BM populations: HSC, CFU-E and MkP, which were sorted as GFP + . B.-E. Venn diagrams representing the overlap between dominant barcodes (top 90% barcodes identified in all PB samples at 12 and 28-weeks post transplantation) detected in anucleate cells (platelets or erythroid cells) and their BM progenitors (MkP or CFU-E) in mouse 1-4

Techniques Used: FACS, Purification, Transplantation Assay

Clonal tracking in blood and bone marrow cells exerts high overlap between detected clones A. UMAP plot showing single cell transcriptomes of sorted BM populations in which barcodes (BC) were detected (all cells which had over 50 000 reads and mitochondrial gene count
Figure Legend Snippet: Clonal tracking in blood and bone marrow cells exerts high overlap between detected clones A. UMAP plot showing single cell transcriptomes of sorted BM populations in which barcodes (BC) were detected (all cells which had over 50 000 reads and mitochondrial gene count

Techniques Used: Clone Assay

A. Gating strategy for PB cell population FACS-purification. Displayed are gates within the MNC/singlet/viable cell population. FACS definitions for purified populations: Mac1 + (CD19 - CD4/8 - CD150 - CD41 - CD45.1 + ), CD19 + (Mac1 - CD4/8 - CD19 + CD150 - CD41 - CD45.1 + ) B. Purity of sorted Mac1 + cells evaluated by FACS Purity of CD19 + cells evaluated by FACS
Figure Legend Snippet: A. Gating strategy for PB cell population FACS-purification. Displayed are gates within the MNC/singlet/viable cell population. FACS definitions for purified populations: Mac1 + (CD19 - CD4/8 - CD150 - CD41 - CD45.1 + ), CD19 + (Mac1 - CD4/8 - CD19 + CD150 - CD41 - CD45.1 + ) B. Purity of sorted Mac1 + cells evaluated by FACS Purity of CD19 + cells evaluated by FACS

Techniques Used: FACS, Purification

In vitro and in vivo validation of RNA barcoding approach for stable, long-term clonal kinetics studies of hematopoiesis A.Experimental set up for in vitro validation of quantitative RNA based clonal studies. The K562 cell line was used for the transduction, single eGFP+ cells were FACS-sorted and expanded to generate monoclonal calibration samples. Four clones were used to create samples, where cells carrying different barcodes were mixed in known ratios-spiked in clone contributing 0.1%, 1%, 5%, 10%, 20% or 50% of total mix. B. Generalised linear model (GLM) correlation between the read count for barcoded transcript retrieved in calibration samples from cDNA or gDNA. C. Experimental setup to test the silencing of eGFP in VavCre x Rosa26tdTomato LSK cells D.-H. eGFP+ chimerism changes in tdTomato+ anucleate cells and CD45.2+ (donor derived) nucleated cells. To calculate the silencing in specific lineage we subtracted eGFP + cells from CD45.2 + cells (nucleated cells-CD19 + , Mac1 + or CD4/8 + cells) or from tdTomato + cells (all donor derived anucleate cells), shown are results from a single experiment in 3 recipient mice
Figure Legend Snippet: In vitro and in vivo validation of RNA barcoding approach for stable, long-term clonal kinetics studies of hematopoiesis A.Experimental set up for in vitro validation of quantitative RNA based clonal studies. The K562 cell line was used for the transduction, single eGFP+ cells were FACS-sorted and expanded to generate monoclonal calibration samples. Four clones were used to create samples, where cells carrying different barcodes were mixed in known ratios-spiked in clone contributing 0.1%, 1%, 5%, 10%, 20% or 50% of total mix. B. Generalised linear model (GLM) correlation between the read count for barcoded transcript retrieved in calibration samples from cDNA or gDNA. C. Experimental setup to test the silencing of eGFP in VavCre x Rosa26tdTomato LSK cells D.-H. eGFP+ chimerism changes in tdTomato+ anucleate cells and CD45.2+ (donor derived) nucleated cells. To calculate the silencing in specific lineage we subtracted eGFP + cells from CD45.2 + cells (nucleated cells-CD19 + , Mac1 + or CD4/8 + cells) or from tdTomato + cells (all donor derived anucleate cells), shown are results from a single experiment in 3 recipient mice

Techniques Used: In Vitro, In Vivo, Transduction, FACS, Clone Assay, Derivative Assay, Mouse Assay

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    Becton Dickinson fluorescence activated cell sorting facs analysis
    Analysis of the Tregs in the MLN and spleen lymphocytes of mice treated with DSS and T. spiralis AES. The lymphocytes isolated from the MLN (A) and spleen (B) of the treated mice were stained with fluorescently conjugated mouse mAbs (anti-CD3e, <t>CD4</t> and CD25), and the intracellular expression of Foxp3 was observed. Representative data from the <t>FACS</t> analysis of the CD4 + CD25 + FOXP3 + Tregs are shown on the left with the percentage of double-positive cells in the right corner. The corresponding bar graphs are shown on the right and indicate the mean percentage ± SE. Significant differences are indicated as asterisks ( P
    Fluorescence Activated Cell Sorting Facs Analysis, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson facs sorted
    Histamine dihydrochloride (HDC)-induced differentiation of leukemic cells is NOX2-dependent. (A) <t>FACS-plots</t> showing NOX2 and H 2 R expression on wild-type (WT) and NOX2 -KO <t>PLB-985</t> cells. Expression of CD11b (B,C) , FPR1 (D) , and FPR2 (E) on WT and NOX2 -KO PLB-985 cells cultured in the presence or absence of HDC or dimethyl sulfoxide (DMSO) as determined by flow cytometry. (F) FACS-plot showing NOX2 and H 2 R expression by OCI-AML3 cells. Expression of CD11b (G) , CD14 (H) , FPR1 (I) , and FPR2 (J) on OCI-AML3 cells cultured in the presence or absence of HDC or DMSO. Abbreviations: MFI, median fluorescence intensity. ANOVA; * p
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    Becton Dickinson facs sorting
    Enrichment of Helios + Treg by sorting on CD103 and GITR. <t>CD4</t> + enriched cells were <t>FACS</t> sorted by gating on CD4 + CD25 + cells, then sorted into three populations based on relative CD103 and GITR staining levels. Data shown are representative of 2 independent experiments, n = 5. A ) Pre and Post sort analysis: Expression of FoxP3 and Helios is shown in both bulk CD4 + CD25 + cells and sorted cells after intracellular staining. B ) qPCR analysis: mRNA was extracted from the FACS sorted populations and reverse transcribed into cDNA. Expression of each mRNA of interest was quantified as compared to an internal control18S rRNA. Data shown are relative expression as compared to a CD4 + CD25 − reference sample. Mean values are plotted +/− SEM.
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    Analysis of the Tregs in the MLN and spleen lymphocytes of mice treated with DSS and T. spiralis AES. The lymphocytes isolated from the MLN (A) and spleen (B) of the treated mice were stained with fluorescently conjugated mouse mAbs (anti-CD3e, CD4 and CD25), and the intracellular expression of Foxp3 was observed. Representative data from the FACS analysis of the CD4 + CD25 + FOXP3 + Tregs are shown on the left with the percentage of double-positive cells in the right corner. The corresponding bar graphs are shown on the right and indicate the mean percentage ± SE. Significant differences are indicated as asterisks ( P

    Journal: PLoS ONE

    Article Title: Excretory/Secretory Products from Trichinella spiralis Adult Worms Ameliorate DSS-Induced Colitis in Mice

    doi: 10.1371/journal.pone.0096454

    Figure Lengend Snippet: Analysis of the Tregs in the MLN and spleen lymphocytes of mice treated with DSS and T. spiralis AES. The lymphocytes isolated from the MLN (A) and spleen (B) of the treated mice were stained with fluorescently conjugated mouse mAbs (anti-CD3e, CD4 and CD25), and the intracellular expression of Foxp3 was observed. Representative data from the FACS analysis of the CD4 + CD25 + FOXP3 + Tregs are shown on the left with the percentage of double-positive cells in the right corner. The corresponding bar graphs are shown on the right and indicate the mean percentage ± SE. Significant differences are indicated as asterisks ( P

    Article Snippet: Fluorescence-activated cell sorting (FACS) analysis of CD4+ CD25+ FOXP3+ Tregs in the spleen and MLN lymphocytes To evaluate the Tregs induced by treatment with the T. spiralis ES products, the isolated cells from the spleens and MLN of the treated mice were sorted by FACS using reagents from BD Pharmingen (USA).

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

    Ferulic acid reduces S-phase cell cycle profiles post to UV treatment. MDA-MB-231 cells were cultured with 10 μM ferulic acid/or DMSO for 24 h. The cells were exposed to UV treatment and harvested. The cell pellets were fixed in 70% ethanol and stained with PI for FACS analysis. UV-; UV untreated, UV+; UV treated (20 mJ/s, harvest post to 3 h).

    Journal: Data in Brief

    Article Title: Data on cell cycle in breast cancer cell line, MDA-MB-231 with ferulic acid treatment

    doi: 10.1016/j.dib.2016.02.001

    Figure Lengend Snippet: Ferulic acid reduces S-phase cell cycle profiles post to UV treatment. MDA-MB-231 cells were cultured with 10 μM ferulic acid/or DMSO for 24 h. The cells were exposed to UV treatment and harvested. The cell pellets were fixed in 70% ethanol and stained with PI for FACS analysis. UV-; UV untreated, UV+; UV treated (20 mJ/s, harvest post to 3 h).

    Article Snippet: 2.2 Cell cycle analysis MDA-MB-231 breast cancer cells were pretreated with ferulic acid or DMSO for 24 h. The cells were exposed to 20 mJ/s UV treatment and harvested post to 3 h. For fluorescence-activated cell sorting (FACS) analysis, MDA-MB-231 cells were fixed overnight at 4C in 70% ethanol, stained with propidium iodine (PI) for 1 h. The cells analyzed for DNA content using a FACS Calibur machine (BD Biosciences).

    Techniques: Multiple Displacement Amplification, Cell Culture, Staining, FACS

    Histamine dihydrochloride (HDC)-induced differentiation of leukemic cells is NOX2-dependent. (A) FACS-plots showing NOX2 and H 2 R expression on wild-type (WT) and NOX2 -KO PLB-985 cells. Expression of CD11b (B,C) , FPR1 (D) , and FPR2 (E) on WT and NOX2 -KO PLB-985 cells cultured in the presence or absence of HDC or dimethyl sulfoxide (DMSO) as determined by flow cytometry. (F) FACS-plot showing NOX2 and H 2 R expression by OCI-AML3 cells. Expression of CD11b (G) , CD14 (H) , FPR1 (I) , and FPR2 (J) on OCI-AML3 cells cultured in the presence or absence of HDC or DMSO. Abbreviations: MFI, median fluorescence intensity. ANOVA; * p

    Journal: Frontiers in Oncology

    Article Title: Anti-Leukemic Properties of Histamine in Monocytic Leukemia: The Role of NOX2

    doi: 10.3389/fonc.2018.00218

    Figure Lengend Snippet: Histamine dihydrochloride (HDC)-induced differentiation of leukemic cells is NOX2-dependent. (A) FACS-plots showing NOX2 and H 2 R expression on wild-type (WT) and NOX2 -KO PLB-985 cells. Expression of CD11b (B,C) , FPR1 (D) , and FPR2 (E) on WT and NOX2 -KO PLB-985 cells cultured in the presence or absence of HDC or dimethyl sulfoxide (DMSO) as determined by flow cytometry. (F) FACS-plot showing NOX2 and H 2 R expression by OCI-AML3 cells. Expression of CD11b (G) , CD14 (H) , FPR1 (I) , and FPR2 (J) on OCI-AML3 cells cultured in the presence or absence of HDC or DMSO. Abbreviations: MFI, median fluorescence intensity. ANOVA; * p

    Article Snippet: Reverse Transcription Quantitative PCR (qPCR) Analysis of HDC-Treated AML Cells Wild-type and NOX2 -KO PLB-985 cells treated by HDC or DMSO for 2 days were FACS-sorted using a three-laser (405, 488, and 633 nm) BD FACSAria II (BD Biosciences).

    Techniques: FACS, Expressing, Cell Culture, Flow Cytometry, Cytometry, Fluorescence

    Histamine dihydrochloride (HDC) facilitates the differentiation of monocytic primary leukemic cells and may be preferentially efficacious in monocytic forms of leukemia. FACS-plots showing live peripheral blood mononuclear cells from representative newly diagnosed patients with (A) FAB-M0 acute myeloid leukemia (AML) with a dominant immature leukemic population (CD34 + CD33 − CD14 − ) and (B) FAB-M4 AML with two distinct populations: an immature blast population (CD34 + CD33 − CD14 − ) and a mature monocytic population (CD34 − CD33 + CD14 + ). The expression of (C) H 2 R, (D) NOX2, (E) FPR1, and (F) FPR2 on primary AML cells [gated as indicated in (A,B) ] and monocytes from healthy donors was determined by flow cytometry. The M5 leukemia is represented by an open circle. One-way ANOVA. (G–I) Median fluorescence intensity as determined by flow cytometry of (G) HLA-DR, (H) FPR1, and (I) FPR2 on live primary monocytic AML cells (FAB: M4/M5) or non-monocytic AML cells (FAB: M0–M2) cultured for 5 days with GM-CSF/IL-4 in the presence or absence of HDC. Wilcoxon matched pair’s test. * p

    Journal: Frontiers in Oncology

    Article Title: Anti-Leukemic Properties of Histamine in Monocytic Leukemia: The Role of NOX2

    doi: 10.3389/fonc.2018.00218

    Figure Lengend Snippet: Histamine dihydrochloride (HDC) facilitates the differentiation of monocytic primary leukemic cells and may be preferentially efficacious in monocytic forms of leukemia. FACS-plots showing live peripheral blood mononuclear cells from representative newly diagnosed patients with (A) FAB-M0 acute myeloid leukemia (AML) with a dominant immature leukemic population (CD34 + CD33 − CD14 − ) and (B) FAB-M4 AML with two distinct populations: an immature blast population (CD34 + CD33 − CD14 − ) and a mature monocytic population (CD34 − CD33 + CD14 + ). The expression of (C) H 2 R, (D) NOX2, (E) FPR1, and (F) FPR2 on primary AML cells [gated as indicated in (A,B) ] and monocytes from healthy donors was determined by flow cytometry. The M5 leukemia is represented by an open circle. One-way ANOVA. (G–I) Median fluorescence intensity as determined by flow cytometry of (G) HLA-DR, (H) FPR1, and (I) FPR2 on live primary monocytic AML cells (FAB: M4/M5) or non-monocytic AML cells (FAB: M0–M2) cultured for 5 days with GM-CSF/IL-4 in the presence or absence of HDC. Wilcoxon matched pair’s test. * p

    Article Snippet: Reverse Transcription Quantitative PCR (qPCR) Analysis of HDC-Treated AML Cells Wild-type and NOX2 -KO PLB-985 cells treated by HDC or DMSO for 2 days were FACS-sorted using a three-laser (405, 488, and 633 nm) BD FACSAria II (BD Biosciences).

    Techniques: FACS, Expressing, Flow Cytometry, Cytometry, Fluorescence, Cell Culture

    Enrichment of Helios + Treg by sorting on CD103 and GITR. CD4 + enriched cells were FACS sorted by gating on CD4 + CD25 + cells, then sorted into three populations based on relative CD103 and GITR staining levels. Data shown are representative of 2 independent experiments, n = 5. A ) Pre and Post sort analysis: Expression of FoxP3 and Helios is shown in both bulk CD4 + CD25 + cells and sorted cells after intracellular staining. B ) qPCR analysis: mRNA was extracted from the FACS sorted populations and reverse transcribed into cDNA. Expression of each mRNA of interest was quantified as compared to an internal control18S rRNA. Data shown are relative expression as compared to a CD4 + CD25 − reference sample. Mean values are plotted +/− SEM.

    Journal: PLoS ONE

    Article Title: Phenotypic and Functional Properties of Helios+ Regulatory T Cells

    doi: 10.1371/journal.pone.0034547

    Figure Lengend Snippet: Enrichment of Helios + Treg by sorting on CD103 and GITR. CD4 + enriched cells were FACS sorted by gating on CD4 + CD25 + cells, then sorted into three populations based on relative CD103 and GITR staining levels. Data shown are representative of 2 independent experiments, n = 5. A ) Pre and Post sort analysis: Expression of FoxP3 and Helios is shown in both bulk CD4 + CD25 + cells and sorted cells after intracellular staining. B ) qPCR analysis: mRNA was extracted from the FACS sorted populations and reverse transcribed into cDNA. Expression of each mRNA of interest was quantified as compared to an internal control18S rRNA. Data shown are relative expression as compared to a CD4 + CD25 − reference sample. Mean values are plotted +/− SEM.

    Article Snippet: Naïve CD4 T cells (CD4+ CD25− CD62Lhi ) were obtained by FACS sorting using a FACSAria II (BD, Franklin Lakes, NJ).

    Techniques: FACS, Staining, Expressing, Real-time Polymerase Chain Reaction