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<t>FACS</t> analysis and immunohistochemistry of xenograft <t>ALDH</t> + 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
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1) 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

2) 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

3) 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

4) 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

5) 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

6) 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

7) 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

8) 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

9) 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

10) 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

11) 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

12) 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

13) 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

14) 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

15) 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

16) 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

17) 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

18) 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

19) 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

20) 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

21) 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

22) 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

23) 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

24) 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

25) 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

26) 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

27) 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

28) 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

29) Product Images from "Uptake and metabolism of arginine impact Plasmodium development in the liver"

Article Title: Uptake and metabolism of arginine impact Plasmodium development in the liver

Journal: Scientific Reports

doi: 10.1038/s41598-017-04424-y

Intracellular polyamine levels are dependent on CAT2A/B function and inhibition of polyamine synthesis by P . berghei decreases hepatic infection. Polyamines were examined by thin-layer chromatography (TLC) of cellular extracts of ( a ) naïve Huh7 cells, ( b ), naïve mouse primary hepatocytes and ( c ) GFP-expressing P . berghei -infected Huh7 cells FACS-sorted at 40 hpi. The amount of each sample was normalized by GAPDH Western blot before analysis. All panels: Top – representative image of polyamines band analyzed by TLC; Bottom – Quantification of band intensities. Representative experiment out of 2 independent experiments. Error bars represent SD of 3 technical replicates. ( d ) Huh7 cells were infected with luciferase-expressing P . berghei sporozoites and the culture medium was replaced by medium with increasing concentrations of DFMO. Parasite load (bioluminescence) and cell viability were assessed at 48 hpi. ( e ) Huh7 cells with the knockdown of ODC and arginase 1 were infected with GFP-expressing P . berghei sporozoites and parasite development was assessed by flow cytometry at 48 hpi. Pool of 3 independent experiments. Error bars represent SEM. ( a ) One-way ANOVA with post-test Dunnett; ( b ) and ( c ) Two-tailed t-test; ( d ) and ( e ) One-way ANOVA with post-test Dunnett. ns - not significant, *p
Figure Legend Snippet: Intracellular polyamine levels are dependent on CAT2A/B function and inhibition of polyamine synthesis by P . berghei decreases hepatic infection. Polyamines were examined by thin-layer chromatography (TLC) of cellular extracts of ( a ) naïve Huh7 cells, ( b ), naïve mouse primary hepatocytes and ( c ) GFP-expressing P . berghei -infected Huh7 cells FACS-sorted at 40 hpi. The amount of each sample was normalized by GAPDH Western blot before analysis. All panels: Top – representative image of polyamines band analyzed by TLC; Bottom – Quantification of band intensities. Representative experiment out of 2 independent experiments. Error bars represent SD of 3 technical replicates. ( d ) Huh7 cells were infected with luciferase-expressing P . berghei sporozoites and the culture medium was replaced by medium with increasing concentrations of DFMO. Parasite load (bioluminescence) and cell viability were assessed at 48 hpi. ( e ) Huh7 cells with the knockdown of ODC and arginase 1 were infected with GFP-expressing P . berghei sporozoites and parasite development was assessed by flow cytometry at 48 hpi. Pool of 3 independent experiments. Error bars represent SEM. ( a ) One-way ANOVA with post-test Dunnett; ( b ) and ( c ) Two-tailed t-test; ( d ) and ( e ) One-way ANOVA with post-test Dunnett. ns - not significant, *p

Techniques Used: Inhibition, Infection, Thin Layer Chromatography, Expressing, FACS, Western Blot, Luciferase, Flow Cytometry, Cytometry, Two Tailed Test

30) Product Images from "ΔN63 suppresses the ability of pregnancy-identified mammary epithelial cells (PIMECs) to drive HER2-positive breast cancer"

Article Title: ΔN63 suppresses the ability of pregnancy-identified mammary epithelial cells (PIMECs) to drive HER2-positive breast cancer

Journal: Cell Death & Disease

doi: 10.1038/s41419-021-03795-5

Downregulation of TAp63 and upregulation of ΔNp63 isoforms in p63+/− luminal cells. a Quantitative RT-PCR on independent p63+/+ ( n = 2) and p63+/− ( n = 3) CD24 high (i.e., luminal) FACS-sorted cells. b – e ScRNA-seq analysis of pooled p63+/+ ( n = 4) and p63+/− ( n = 3) CD24 pos (i.e., mammary epithelial) cells. b UMAP plots visualized in Loupe browser, see Supplementary Table 2 for details. c Krt8 marks luminal cells. d , e Average expression ( d ) and representative UMAP plots ( e ) of significantly altered TAp63 and ΔNp63 target genes in the luminal cluster of p63+/+ and p63+/− mammary epithelial cells, ** p
Figure Legend Snippet: Downregulation of TAp63 and upregulation of ΔNp63 isoforms in p63+/− luminal cells. a Quantitative RT-PCR on independent p63+/+ ( n = 2) and p63+/− ( n = 3) CD24 high (i.e., luminal) FACS-sorted cells. b – e ScRNA-seq analysis of pooled p63+/+ ( n = 4) and p63+/− ( n = 3) CD24 pos (i.e., mammary epithelial) cells. b UMAP plots visualized in Loupe browser, see Supplementary Table 2 for details. c Krt8 marks luminal cells. d , e Average expression ( d ) and representative UMAP plots ( e ) of significantly altered TAp63 and ΔNp63 target genes in the luminal cluster of p63+/+ and p63+/− mammary epithelial cells, ** p

Techniques Used: Quantitative RT-PCR, FACS, Expressing

31) 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

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

32) Product Images from "Trajectory Mapping of the Early Drosophila Germline Reveals Controls of Zygotic Activation and Sex Differentiation"

Article Title: Trajectory Mapping of the Early Drosophila Germline Reveals Controls of Zygotic Activation and Sex Differentiation

Journal: bioRxiv

doi: 10.1101/2020.09.11.292573

FACS schemes for the collection of single germ cells for scRNA-seq. a-b , FACS plots for unsexed 0-4 h germ cells. c-d , FACS plots for unsexed 4-8 germ cells. a, c , Gating for live cells which plots FSC-SSC. b, d , The gate for the magenta cells indicate the GFP+ cells that were sorted and their percentages. X and Y axes indicate the FITC and PE channels, respectively. e , Female and male 5-8 h embryos can be separately based on their Sxl-GFP expression under a fluorescent stereoscope. The embryos in the upper half express GFP highly and are females; those in the lower half are males. f-g , FACS plots for female 5-8 h germ cells. h-i , FACS plots for male 5-8 h germ cells. f, h , The small encircled populations on the bottom of the plots are enriched for germ cells, thus this live gate was used for the FITC-PE plots ( g, i ). g, i , FITC-PE plots with magenta cells being the GFP+ cells sorted. The axes are the same as in b, d .
Figure Legend Snippet: FACS schemes for the collection of single germ cells for scRNA-seq. a-b , FACS plots for unsexed 0-4 h germ cells. c-d , FACS plots for unsexed 4-8 germ cells. a, c , Gating for live cells which plots FSC-SSC. b, d , The gate for the magenta cells indicate the GFP+ cells that were sorted and their percentages. X and Y axes indicate the FITC and PE channels, respectively. e , Female and male 5-8 h embryos can be separately based on their Sxl-GFP expression under a fluorescent stereoscope. The embryos in the upper half express GFP highly and are females; those in the lower half are males. f-g , FACS plots for female 5-8 h germ cells. h-i , FACS plots for male 5-8 h germ cells. f, h , The small encircled populations on the bottom of the plots are enriched for germ cells, thus this live gate was used for the FITC-PE plots ( g, i ). g, i , FITC-PE plots with magenta cells being the GFP+ cells sorted. The axes are the same as in b, d .

Techniques Used: FACS, Expressing

Clustering and pseudotime analysis of single-cell germline transcriptomes. a , FACS plot for sorting germ cells from embryonic homogenates of 0-8 h embryos with the GFP+ germ cells highlighted by a dotted circle. X-axis is the green channel. b , Morphology by DIC and GFP profile of sorted 0-8 h germ cells in the unsexed sample. Note that virtually all cells recovered are GFP-positive cells whose morphology resemble that of germline. c , Clustering analysis of the unsexed sample. Each dot represents a cell in the dataset, and individual clusters are numbered and colored differently. d , Expression profile of nos of the unsexed sample showing that clusters 1 and 2 make up the main germline cluster. Each dot represents a cell in the dataset, the numbers indicate individual clusters, and the color code for expression levels is indicated on the right. e , Pseudotime analysis of the germline cluster from the unsexed sample. The black lines within the clusters indicate pseudotime trajectories. The legend on the right explains the color code of pseudotime. f , The sexed dataset is plotted after dimension reduction. Each blue and magenta dot represents a cell from the male and female dataset, respectively. g , Clustering analysis of the sexed sample. Each dot represents a cell in the dataset, and individual clusters are numbered and colored differently. h , Expression profile of nos after clustering analysis of the sexed dataset indicating that clusters 1, 7, and 8 contain germ cells. Details of the graph is the same as in b. i , Pseudotime analysis of the germline cluster from the sexed dataset. Black lines indicate pseudotime trajectories and the color codes of pseudotime is indicated on the right.
Figure Legend Snippet: Clustering and pseudotime analysis of single-cell germline transcriptomes. a , FACS plot for sorting germ cells from embryonic homogenates of 0-8 h embryos with the GFP+ germ cells highlighted by a dotted circle. X-axis is the green channel. b , Morphology by DIC and GFP profile of sorted 0-8 h germ cells in the unsexed sample. Note that virtually all cells recovered are GFP-positive cells whose morphology resemble that of germline. c , Clustering analysis of the unsexed sample. Each dot represents a cell in the dataset, and individual clusters are numbered and colored differently. d , Expression profile of nos of the unsexed sample showing that clusters 1 and 2 make up the main germline cluster. Each dot represents a cell in the dataset, the numbers indicate individual clusters, and the color code for expression levels is indicated on the right. e , Pseudotime analysis of the germline cluster from the unsexed sample. The black lines within the clusters indicate pseudotime trajectories. The legend on the right explains the color code of pseudotime. f , The sexed dataset is plotted after dimension reduction. Each blue and magenta dot represents a cell from the male and female dataset, respectively. g , Clustering analysis of the sexed sample. Each dot represents a cell in the dataset, and individual clusters are numbered and colored differently. h , Expression profile of nos after clustering analysis of the sexed dataset indicating that clusters 1, 7, and 8 contain germ cells. Details of the graph is the same as in b. i , Pseudotime analysis of the germline cluster from the sexed dataset. Black lines indicate pseudotime trajectories and the color codes of pseudotime is indicated on the right.

Techniques Used: FACS, Expressing

33) Product Images from "PD-1 is a haploinsufficient suppressor of T cell lymphomagenesis"

Article Title: PD-1 is a haploinsufficient suppressor of T cell lymphomagenesis

Journal: Nature

doi: 10.1038/nature24649

Lymphomas with transposon insertions within Pdcd1. a, Overall survival of the Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre mice (red) and littermate ITK-SYK CD4-Cre mice (blue). The Rosa26 LSL-PB ;ATP2;CD4-Cre mice (black) did not display any signs of disease. The p-value was determined using two-sided log-rank test, and the median survival was 255 days vs. 289 days. b, Flow cytometric analyses of TCRβ, CD4 and eGFP expression in a typical lymphoma sample from a Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre mouse. c, Histological analysis of the mouse shown in b with multinodal perivascular infiltrations of lymphoblasts into the liver (left) and lung (right). Scale bars represent 200 µm and 20 µm (inset). d, Pdcd1 mRNA transcript levels in eGFP + FACS-sorted peripheral lymphocytes from Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre lymphoma samples without any detectable transposon cassettes within the Pdcd1 locus (None, n=4) or with a single insertion (Single, n=4) or multiple Pdcd1 -located transposon cassettes (Multiple, n=4). Pdcd1 expression levels were normalized to eGFP transcript levels. P=Tukey’s post hoc test. Shown are the mean±s.e.m and individual data points. e, Twenty unique transposon integration sites mapped to the murine Pdcd1 locus. The vertical black bars indicate the genomic positions of the insertion. The red arrows indicate the orientations of the transposon cassettes. f , Nucleotide sequences of the Pdcd1 - piggyBac mRNA splice junctions as determined by Sanger sequencing of amplified cDNA from Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre lymphoma samples. The cDNA was generated from sorted eGFP + lymphoma cells in which at least one Pdcd1 transposon cassette could be detected. Note that the 5’ piggyBac transposon-specific inverted terminal repeat nucleotide sequence (PB5) can function as a cryptic splice acceptor and a splice donor site. Hence, PB5 may act as a gene trapping sequence by itself 14 . En2SA, exon 2 located murine Engrailed 2 splice acceptor; pA, SV40 polyadenylation signal; MSCV, murine stem cell virus long terminal repeat; LunSD, splice donor from exon 1 of murine Foxf2. b, c, Representative data from at least ten analysed mice. f , The splice junctions were amplified from the cDNA of at least eight lymphomas.
Figure Legend Snippet: Lymphomas with transposon insertions within Pdcd1. a, Overall survival of the Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre mice (red) and littermate ITK-SYK CD4-Cre mice (blue). The Rosa26 LSL-PB ;ATP2;CD4-Cre mice (black) did not display any signs of disease. The p-value was determined using two-sided log-rank test, and the median survival was 255 days vs. 289 days. b, Flow cytometric analyses of TCRβ, CD4 and eGFP expression in a typical lymphoma sample from a Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre mouse. c, Histological analysis of the mouse shown in b with multinodal perivascular infiltrations of lymphoblasts into the liver (left) and lung (right). Scale bars represent 200 µm and 20 µm (inset). d, Pdcd1 mRNA transcript levels in eGFP + FACS-sorted peripheral lymphocytes from Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre lymphoma samples without any detectable transposon cassettes within the Pdcd1 locus (None, n=4) or with a single insertion (Single, n=4) or multiple Pdcd1 -located transposon cassettes (Multiple, n=4). Pdcd1 expression levels were normalized to eGFP transcript levels. P=Tukey’s post hoc test. Shown are the mean±s.e.m and individual data points. e, Twenty unique transposon integration sites mapped to the murine Pdcd1 locus. The vertical black bars indicate the genomic positions of the insertion. The red arrows indicate the orientations of the transposon cassettes. f , Nucleotide sequences of the Pdcd1 - piggyBac mRNA splice junctions as determined by Sanger sequencing of amplified cDNA from Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre lymphoma samples. The cDNA was generated from sorted eGFP + lymphoma cells in which at least one Pdcd1 transposon cassette could be detected. Note that the 5’ piggyBac transposon-specific inverted terminal repeat nucleotide sequence (PB5) can function as a cryptic splice acceptor and a splice donor site. Hence, PB5 may act as a gene trapping sequence by itself 14 . En2SA, exon 2 located murine Engrailed 2 splice acceptor; pA, SV40 polyadenylation signal; MSCV, murine stem cell virus long terminal repeat; LunSD, splice donor from exon 1 of murine Foxf2. b, c, Representative data from at least ten analysed mice. f , The splice junctions were amplified from the cDNA of at least eight lymphomas.

Techniques Used: Mouse Assay, Flow Cytometry, Expressing, FACS, Sequencing, Amplification, Generated, Activated Clotting Time Assay

Identification of PDCD1 alterations in T cell lymphoma. a, Schematic representation of the transposon screen to discover T cell lymphoma genes . Transposons from the ATP2 transgene were mobilized by piggyBac in ITK-SYK-expressing T cells. The tumour cells were then FACS-sorted based on eGFP expression, and the transposon insertion sites were identified by next-generation sequencing and bioinformatics analysis. b, Transposon insertion densities within pooled lymphomas (n=30 mice) for the murine chromosome 1, total: 2732 insertions and Pdcd1 locus: 23 insertions. The p-value was calculated with a one-sided Poisson distribution based test, which assumes transposon insertions occurring at a constant rate. c, Flow cytometric analysis of PD-1 expression on lymphoma cells from diseased Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre mice. Lymphoma sample without any transposon cassette within the Pdcd1 gene (No insertion), with a single transposon cassette located in the Pdcd1 locus (Single insertion) or with multiple transposon cassettes within Pdcd1 (Multiple insertions). d, The dashed box indicates the genomic region q37.3 on human chromosome 2 that is shown in detail in the lower part of the panel (top). Mono- or bi-allelic deletions of the PDCD1 gene detected in human T cell lymphoma patients are shown (bottom). The horizontal bars indicate the regions that were affected by the CNAs. The colour of the bar indicates the type of CNA. CNA, copy number aberration; Mbp, million base pairs c, Representative results from at least 15 mice. d, Results of an analysis that included all available WGS data within one published dataset.
Figure Legend Snippet: Identification of PDCD1 alterations in T cell lymphoma. a, Schematic representation of the transposon screen to discover T cell lymphoma genes . Transposons from the ATP2 transgene were mobilized by piggyBac in ITK-SYK-expressing T cells. The tumour cells were then FACS-sorted based on eGFP expression, and the transposon insertion sites were identified by next-generation sequencing and bioinformatics analysis. b, Transposon insertion densities within pooled lymphomas (n=30 mice) for the murine chromosome 1, total: 2732 insertions and Pdcd1 locus: 23 insertions. The p-value was calculated with a one-sided Poisson distribution based test, which assumes transposon insertions occurring at a constant rate. c, Flow cytometric analysis of PD-1 expression on lymphoma cells from diseased Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre mice. Lymphoma sample without any transposon cassette within the Pdcd1 gene (No insertion), with a single transposon cassette located in the Pdcd1 locus (Single insertion) or with multiple transposon cassettes within Pdcd1 (Multiple insertions). d, The dashed box indicates the genomic region q37.3 on human chromosome 2 that is shown in detail in the lower part of the panel (top). Mono- or bi-allelic deletions of the PDCD1 gene detected in human T cell lymphoma patients are shown (bottom). The horizontal bars indicate the regions that were affected by the CNAs. The colour of the bar indicates the type of CNA. CNA, copy number aberration; Mbp, million base pairs c, Representative results from at least 15 mice. d, Results of an analysis that included all available WGS data within one published dataset.

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

PD-1 is a haploinsufficient tumour suppressor in vivo . a, ITK-SYK CD4-CreERT2 and ITK-SYK CD4-CreERT2 ;PD-1 -/- mice (n=3 mice per genotype) received single tamoxifen injections (0.25 mg). The percentages of ITK-SYK-expressing eGFP + blood lymphocytes were determined using flow cytometry. b , Fifty million splenic cells from diseased ITK-SYK CD4-CreERT2 ;PD-1 -/- mice (n=3 donors) were intravenously transferred to NSG mice (n=3 recipients). The percentage of eGFP + lymphocytes in the recipient’s blood is indicated. NSG, NOD SCID Il2rg −/− c , Survival of ITK-SYK CD4-CreERT2 , ITK-SYK CD4-CreERT2 ;PD-1 +/- and ITK-SYK CD4-CreERT2 ;PD-1 -/- mice (n=6, n=12 and n=14). All mice received a single tamoxifen dose on day zero. P=two-sided log-rank test d , The percentages of ITK-SYK-expressing eGFP + peripheral blood lymphocytes of checkpoint inhibitor-treated ITK-SYK CD4-CreERT2 mice were determined by FACS. ITK-SYK CD4-CreERT2 mice received single tamoxifen injections. Beginning twenty-four hours later, the mice received 200 µg of anti-PD-L1 or control antibody (n=3 mice per condition) every second day. e, Anti-CD3 antibody-stained liver section from a diseased anti-PD-L1-treated ITK-SYK CD4-CreERT2 mouse from the experiment shown in d , Scale bars represent 100 µm and 20 µm (inset). f, Survival of ITK-SYK CD4-CreERT2 mice that received checkpoint inhibitor treatment starting ten days after tamoxifen administration. ITK-SYK CD4-CreERT2 mice received single injections of tamoxifen on day zero (0.25 mg). Starting ten days after injection, mice were administered 200 µg of anti-PD-L1 or anti-PD-1 antibody or control antibody (n=4 mice per condition) every third day. P=two-sided log-rank test g, Phosflow analyses of PTEN, p-AKT and p-PKCθ levels in ITK-SYK-expressing T cells isolated from the C57BL/6 recipients presented in Extended Data Figure 6b . The genotype of the transplanted cells is indicated. h, Survival of the PI3Kδ inhibitor- or vehicle-treated recipient mice. Tamoxifen-induced ITK-SYK CD4-CreERT2 ;PD-1 -/- T cells (n=5 biological replicates, 5x10 4 cells per transplant) were transplanted into NSG mice (n=5 recipients per group). P=two-sided log-rank test. † indicates animals that were euthanized because of lymphomas. a, Representative data from three independent experiments, each with three biological replicates. b, The data from a single experiment that was independently repeated once with comparable results. c , Pooled data from two independent experiments. d , Representative data from four independent experiments, each with three biological replicates per antibody. e , Representative histology from one out of nine analysed mice. f, Data from a single experiment, which was repeated once with similar results. g, Representative results from two out of eight analysed mice in two independent experiments. h, Pooled survival data from two experiments.
Figure Legend Snippet: PD-1 is a haploinsufficient tumour suppressor in vivo . a, ITK-SYK CD4-CreERT2 and ITK-SYK CD4-CreERT2 ;PD-1 -/- mice (n=3 mice per genotype) received single tamoxifen injections (0.25 mg). The percentages of ITK-SYK-expressing eGFP + blood lymphocytes were determined using flow cytometry. b , Fifty million splenic cells from diseased ITK-SYK CD4-CreERT2 ;PD-1 -/- mice (n=3 donors) were intravenously transferred to NSG mice (n=3 recipients). The percentage of eGFP + lymphocytes in the recipient’s blood is indicated. NSG, NOD SCID Il2rg −/− c , Survival of ITK-SYK CD4-CreERT2 , ITK-SYK CD4-CreERT2 ;PD-1 +/- and ITK-SYK CD4-CreERT2 ;PD-1 -/- mice (n=6, n=12 and n=14). All mice received a single tamoxifen dose on day zero. P=two-sided log-rank test d , The percentages of ITK-SYK-expressing eGFP + peripheral blood lymphocytes of checkpoint inhibitor-treated ITK-SYK CD4-CreERT2 mice were determined by FACS. ITK-SYK CD4-CreERT2 mice received single tamoxifen injections. Beginning twenty-four hours later, the mice received 200 µg of anti-PD-L1 or control antibody (n=3 mice per condition) every second day. e, Anti-CD3 antibody-stained liver section from a diseased anti-PD-L1-treated ITK-SYK CD4-CreERT2 mouse from the experiment shown in d , Scale bars represent 100 µm and 20 µm (inset). f, Survival of ITK-SYK CD4-CreERT2 mice that received checkpoint inhibitor treatment starting ten days after tamoxifen administration. ITK-SYK CD4-CreERT2 mice received single injections of tamoxifen on day zero (0.25 mg). Starting ten days after injection, mice were administered 200 µg of anti-PD-L1 or anti-PD-1 antibody or control antibody (n=4 mice per condition) every third day. P=two-sided log-rank test g, Phosflow analyses of PTEN, p-AKT and p-PKCθ levels in ITK-SYK-expressing T cells isolated from the C57BL/6 recipients presented in Extended Data Figure 6b . The genotype of the transplanted cells is indicated. h, Survival of the PI3Kδ inhibitor- or vehicle-treated recipient mice. Tamoxifen-induced ITK-SYK CD4-CreERT2 ;PD-1 -/- T cells (n=5 biological replicates, 5x10 4 cells per transplant) were transplanted into NSG mice (n=5 recipients per group). P=two-sided log-rank test. † indicates animals that were euthanized because of lymphomas. a, Representative data from three independent experiments, each with three biological replicates. b, The data from a single experiment that was independently repeated once with comparable results. c , Pooled data from two independent experiments. d , Representative data from four independent experiments, each with three biological replicates per antibody. e , Representative histology from one out of nine analysed mice. f, Data from a single experiment, which was repeated once with similar results. g, Representative results from two out of eight analysed mice in two independent experiments. h, Pooled survival data from two experiments.

Techniques Used: In Vivo, Mouse Assay, Expressing, Flow Cytometry, Cytometry, FACS, Staining, Injection, Isolation

34) Product Images from "Glucocorticoids Regulate Bone Marrow B Lymphopoiesis After Stroke"

Article Title: Glucocorticoids Regulate Bone Marrow B Lymphopoiesis After Stroke

Journal: Circulation research

doi: 10.1161/CIRCRESAHA.118.314518

B cell progenitor loss in the bone marrow does not result in mobilization. A , Representative FACS staining of early B cell progenitors (lineage − B220 int CD93 + cells) in bone marrow, blood, and spleen at indicated time points after tMCAO. B , FACS-based enumeration of CD19 + B-lymphocytes in blood and spleen at indicated days after tMCAO induction. C , Bar graph shows total numbers of cells in the spleen over a three-day period after stroke in mice (n= 6 per group). Mean ± s.e.m; * P
Figure Legend Snippet: B cell progenitor loss in the bone marrow does not result in mobilization. A , Representative FACS staining of early B cell progenitors (lineage − B220 int CD93 + cells) in bone marrow, blood, and spleen at indicated time points after tMCAO. B , FACS-based enumeration of CD19 + B-lymphocytes in blood and spleen at indicated days after tMCAO induction. C , Bar graph shows total numbers of cells in the spleen over a three-day period after stroke in mice (n= 6 per group). Mean ± s.e.m; * P

Techniques Used: FACS, Staining, Mouse Assay

35) Product Images from "PD-1 is a haploinsufficient suppressor of T cell lymphomagenesis"

Article Title: PD-1 is a haploinsufficient suppressor of T cell lymphomagenesis

Journal: Nature

doi: 10.1038/nature24649

Lymphomas with transposon insertions within Pdcd1. a, Overall survival of the Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre mice (red) and littermate ITK-SYK CD4-Cre mice (blue). The Rosa26 LSL-PB ;ATP2;CD4-Cre mice (black) did not display any signs of disease. The p-value was determined using two-sided log-rank test, and the median survival was 255 days vs. 289 days. b, Flow cytometric analyses of TCRβ, CD4 and eGFP expression in a typical lymphoma sample from a Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre mouse. c, Histological analysis of the mouse shown in b with multinodal perivascular infiltrations of lymphoblasts into the liver (left) and lung (right). Scale bars represent 200 µm and 20 µm (inset). d, Pdcd1 mRNA transcript levels in eGFP + FACS-sorted peripheral lymphocytes from Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre lymphoma samples without any detectable transposon cassettes within the Pdcd1 locus (None, n=4) or with a single insertion (Single, n=4) or multiple Pdcd1 -located transposon cassettes (Multiple, n=4). Pdcd1 expression levels were normalized to eGFP transcript levels. P=Tukey’s post hoc test. Shown are the mean±s.e.m and individual data points. e, Twenty unique transposon integration sites mapped to the murine Pdcd1 locus. The vertical black bars indicate the genomic positions of the insertion. The red arrows indicate the orientations of the transposon cassettes. f , Nucleotide sequences of the Pdcd1 - piggyBac mRNA splice junctions as determined by Sanger sequencing of amplified cDNA from Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre lymphoma samples. The cDNA was generated from sorted eGFP + lymphoma cells in which at least one Pdcd1 transposon cassette could be detected. Note that the 5’ piggyBac transposon-specific inverted terminal repeat nucleotide sequence (PB5) can function as a cryptic splice acceptor and a splice donor site. Hence, PB5 may act as a gene trapping sequence by itself 14 . En2SA, exon 2 located murine Engrailed 2 splice acceptor; pA, SV40 polyadenylation signal; MSCV, murine stem cell virus long terminal repeat; LunSD, splice donor from exon 1 of murine Foxf2. b, c, Representative data from at least ten analysed mice. f , The splice junctions were amplified from the cDNA of at least eight lymphomas.
Figure Legend Snippet: Lymphomas with transposon insertions within Pdcd1. a, Overall survival of the Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre mice (red) and littermate ITK-SYK CD4-Cre mice (blue). The Rosa26 LSL-PB ;ATP2;CD4-Cre mice (black) did not display any signs of disease. The p-value was determined using two-sided log-rank test, and the median survival was 255 days vs. 289 days. b, Flow cytometric analyses of TCRβ, CD4 and eGFP expression in a typical lymphoma sample from a Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre mouse. c, Histological analysis of the mouse shown in b with multinodal perivascular infiltrations of lymphoblasts into the liver (left) and lung (right). Scale bars represent 200 µm and 20 µm (inset). d, Pdcd1 mRNA transcript levels in eGFP + FACS-sorted peripheral lymphocytes from Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre lymphoma samples without any detectable transposon cassettes within the Pdcd1 locus (None, n=4) or with a single insertion (Single, n=4) or multiple Pdcd1 -located transposon cassettes (Multiple, n=4). Pdcd1 expression levels were normalized to eGFP transcript levels. P=Tukey’s post hoc test. Shown are the mean±s.e.m and individual data points. e, Twenty unique transposon integration sites mapped to the murine Pdcd1 locus. The vertical black bars indicate the genomic positions of the insertion. The red arrows indicate the orientations of the transposon cassettes. f , Nucleotide sequences of the Pdcd1 - piggyBac mRNA splice junctions as determined by Sanger sequencing of amplified cDNA from Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre lymphoma samples. The cDNA was generated from sorted eGFP + lymphoma cells in which at least one Pdcd1 transposon cassette could be detected. Note that the 5’ piggyBac transposon-specific inverted terminal repeat nucleotide sequence (PB5) can function as a cryptic splice acceptor and a splice donor site. Hence, PB5 may act as a gene trapping sequence by itself 14 . En2SA, exon 2 located murine Engrailed 2 splice acceptor; pA, SV40 polyadenylation signal; MSCV, murine stem cell virus long terminal repeat; LunSD, splice donor from exon 1 of murine Foxf2. b, c, Representative data from at least ten analysed mice. f , The splice junctions were amplified from the cDNA of at least eight lymphomas.

Techniques Used: Mouse Assay, Flow Cytometry, Expressing, FACS, Sequencing, Amplification, Generated, Activated Clotting Time Assay

Identification of PDCD1 alterations in T cell lymphoma. a, Schematic representation of the transposon screen to discover T cell lymphoma genes . Transposons from the ATP2 transgene were mobilized by piggyBac in ITK-SYK-expressing T cells. The tumour cells were then FACS-sorted based on eGFP expression, and the transposon insertion sites were identified by next-generation sequencing and bioinformatics analysis. b, Transposon insertion densities within pooled lymphomas (n=30 mice) for the murine chromosome 1, total: 2732 insertions and Pdcd1 locus: 23 insertions. The p-value was calculated with a one-sided Poisson distribution based test, which assumes transposon insertions occurring at a constant rate. c, Flow cytometric analysis of PD-1 expression on lymphoma cells from diseased Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre mice. Lymphoma sample without any transposon cassette within the Pdcd1 gene (No insertion), with a single transposon cassette located in the Pdcd1 locus (Single insertion) or with multiple transposon cassettes within Pdcd1 (Multiple insertions). d, The dashed box indicates the genomic region q37.3 on human chromosome 2 that is shown in detail in the lower part of the panel (top). Mono- or bi-allelic deletions of the PDCD1 gene detected in human T cell lymphoma patients are shown (bottom). The horizontal bars indicate the regions that were affected by the CNAs. The colour of the bar indicates the type of CNA. CNA, copy number aberration; Mbp, million base pairs c, Representative results from at least 15 mice. d, Results of an analysis that included all available WGS data within one published dataset.
Figure Legend Snippet: Identification of PDCD1 alterations in T cell lymphoma. a, Schematic representation of the transposon screen to discover T cell lymphoma genes . Transposons from the ATP2 transgene were mobilized by piggyBac in ITK-SYK-expressing T cells. The tumour cells were then FACS-sorted based on eGFP expression, and the transposon insertion sites were identified by next-generation sequencing and bioinformatics analysis. b, Transposon insertion densities within pooled lymphomas (n=30 mice) for the murine chromosome 1, total: 2732 insertions and Pdcd1 locus: 23 insertions. The p-value was calculated with a one-sided Poisson distribution based test, which assumes transposon insertions occurring at a constant rate. c, Flow cytometric analysis of PD-1 expression on lymphoma cells from diseased Rosa26 LSL-ITK-SYK ;Rosa26 LSL-PB ;ATP2;CD4-Cre mice. Lymphoma sample without any transposon cassette within the Pdcd1 gene (No insertion), with a single transposon cassette located in the Pdcd1 locus (Single insertion) or with multiple transposon cassettes within Pdcd1 (Multiple insertions). d, The dashed box indicates the genomic region q37.3 on human chromosome 2 that is shown in detail in the lower part of the panel (top). Mono- or bi-allelic deletions of the PDCD1 gene detected in human T cell lymphoma patients are shown (bottom). The horizontal bars indicate the regions that were affected by the CNAs. The colour of the bar indicates the type of CNA. CNA, copy number aberration; Mbp, million base pairs c, Representative results from at least 15 mice. d, Results of an analysis that included all available WGS data within one published dataset.

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

PD-1 is a haploinsufficient tumour suppressor in vivo . a, ITK-SYK CD4-CreERT2 and ITK-SYK CD4-CreERT2 ;PD-1 -/- mice (n=3 mice per genotype) received single tamoxifen injections (0.25 mg). The percentages of ITK-SYK-expressing eGFP + blood lymphocytes were determined using flow cytometry. b , Fifty million splenic cells from diseased ITK-SYK CD4-CreERT2 ;PD-1 -/- mice (n=3 donors) were intravenously transferred to NSG mice (n=3 recipients). The percentage of eGFP + lymphocytes in the recipient’s blood is indicated. NSG, NOD SCID Il2rg −/− c , Survival of ITK-SYK CD4-CreERT2 , ITK-SYK CD4-CreERT2 ;PD-1 +/- and ITK-SYK CD4-CreERT2 ;PD-1 -/- mice (n=6, n=12 and n=14). All mice received a single tamoxifen dose on day zero. P=two-sided log-rank test d , The percentages of ITK-SYK-expressing eGFP + peripheral blood lymphocytes of checkpoint inhibitor-treated ITK-SYK CD4-CreERT2 mice were determined by FACS. ITK-SYK CD4-CreERT2 mice received single tamoxifen injections. Beginning twenty-four hours later, the mice received 200 µg of anti-PD-L1 or control antibody (n=3 mice per condition) every second day. e, Anti-CD3 antibody-stained liver section from a diseased anti-PD-L1-treated ITK-SYK CD4-CreERT2 mouse from the experiment shown in d , Scale bars represent 100 µm and 20 µm (inset). f, Survival of ITK-SYK CD4-CreERT2 mice that received checkpoint inhibitor treatment starting ten days after tamoxifen administration. ITK-SYK CD4-CreERT2 mice received single injections of tamoxifen on day zero (0.25 mg). Starting ten days after injection, mice were administered 200 µg of anti-PD-L1 or anti-PD-1 antibody or control antibody (n=4 mice per condition) every third day. P=two-sided log-rank test g, Phosflow analyses of PTEN, p-AKT and p-PKCθ levels in ITK-SYK-expressing T cells isolated from the C57BL/6 recipients presented in Extended Data Figure 6b . The genotype of the transplanted cells is indicated. h, Survival of the PI3Kδ inhibitor- or vehicle-treated recipient mice. Tamoxifen-induced ITK-SYK CD4-CreERT2 ;PD-1 -/- T cells (n=5 biological replicates, 5x10 4 cells per transplant) were transplanted into NSG mice (n=5 recipients per group). P=two-sided log-rank test. † indicates animals that were euthanized because of lymphomas. a, Representative data from three independent experiments, each with three biological replicates. b, The data from a single experiment that was independently repeated once with comparable results. c , Pooled data from two independent experiments. d , Representative data from four independent experiments, each with three biological replicates per antibody. e , Representative histology from one out of nine analysed mice. f, Data from a single experiment, which was repeated once with similar results. g, Representative results from two out of eight analysed mice in two independent experiments. h, Pooled survival data from two experiments.
Figure Legend Snippet: PD-1 is a haploinsufficient tumour suppressor in vivo . a, ITK-SYK CD4-CreERT2 and ITK-SYK CD4-CreERT2 ;PD-1 -/- mice (n=3 mice per genotype) received single tamoxifen injections (0.25 mg). The percentages of ITK-SYK-expressing eGFP + blood lymphocytes were determined using flow cytometry. b , Fifty million splenic cells from diseased ITK-SYK CD4-CreERT2 ;PD-1 -/- mice (n=3 donors) were intravenously transferred to NSG mice (n=3 recipients). The percentage of eGFP + lymphocytes in the recipient’s blood is indicated. NSG, NOD SCID Il2rg −/− c , Survival of ITK-SYK CD4-CreERT2 , ITK-SYK CD4-CreERT2 ;PD-1 +/- and ITK-SYK CD4-CreERT2 ;PD-1 -/- mice (n=6, n=12 and n=14). All mice received a single tamoxifen dose on day zero. P=two-sided log-rank test d , The percentages of ITK-SYK-expressing eGFP + peripheral blood lymphocytes of checkpoint inhibitor-treated ITK-SYK CD4-CreERT2 mice were determined by FACS. ITK-SYK CD4-CreERT2 mice received single tamoxifen injections. Beginning twenty-four hours later, the mice received 200 µg of anti-PD-L1 or control antibody (n=3 mice per condition) every second day. e, Anti-CD3 antibody-stained liver section from a diseased anti-PD-L1-treated ITK-SYK CD4-CreERT2 mouse from the experiment shown in d , Scale bars represent 100 µm and 20 µm (inset). f, Survival of ITK-SYK CD4-CreERT2 mice that received checkpoint inhibitor treatment starting ten days after tamoxifen administration. ITK-SYK CD4-CreERT2 mice received single injections of tamoxifen on day zero (0.25 mg). Starting ten days after injection, mice were administered 200 µg of anti-PD-L1 or anti-PD-1 antibody or control antibody (n=4 mice per condition) every third day. P=two-sided log-rank test g, Phosflow analyses of PTEN, p-AKT and p-PKCθ levels in ITK-SYK-expressing T cells isolated from the C57BL/6 recipients presented in Extended Data Figure 6b . The genotype of the transplanted cells is indicated. h, Survival of the PI3Kδ inhibitor- or vehicle-treated recipient mice. Tamoxifen-induced ITK-SYK CD4-CreERT2 ;PD-1 -/- T cells (n=5 biological replicates, 5x10 4 cells per transplant) were transplanted into NSG mice (n=5 recipients per group). P=two-sided log-rank test. † indicates animals that were euthanized because of lymphomas. a, Representative data from three independent experiments, each with three biological replicates. b, The data from a single experiment that was independently repeated once with comparable results. c , Pooled data from two independent experiments. d , Representative data from four independent experiments, each with three biological replicates per antibody. e , Representative histology from one out of nine analysed mice. f, Data from a single experiment, which was repeated once with similar results. g, Representative results from two out of eight analysed mice in two independent experiments. h, Pooled survival data from two experiments.

Techniques Used: In Vivo, Mouse Assay, Expressing, Flow Cytometry, Cytometry, FACS, Staining, Injection, Isolation

36) Product Images from "An Immunocompetent Mouse Model for MLL/AF9 Leukemia Reveals the Potential of Spontaneous Cytotoxic T-Cell Response to an Antigen Expressed in Leukemia Cells"

Article Title: An Immunocompetent Mouse Model for MLL/AF9 Leukemia Reveals the Potential of Spontaneous Cytotoxic T-Cell Response to an Antigen Expressed in Leukemia Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0144594

Spontaneous regression of leukemia was observed in the presence, but not in the absence, of adaptive immunity. (A) Flow-cytometry analyses of BM cells of non-irradiated wild-type recipients 7 days after transplantation with different numbers (3 × 10 3 , 3 × 10 4 , or 3 × 10 5 ) of MLL/AF9-OVA leukemia cells. (B) FACS analysis of BM from non-irradiated wild-type or Rag2 -/- mice transplanted with 3 × 10 4 MLL/AF9-OVA leukemia cells. Mice were analyzed 3 weeks after transplant. (C) Kaplan–Meier curves for overall survival of non-irradiated wild-type (n = 7) or Rag2 -/- (n = 3) recipients transplanted with 3 × 10 4 MLL/AF9-OVA leukemia cells. (D) Percentages of GFP + leukemia cells in BM after transplantation into non-irradiated wild-type mice were examined every week. Each dot and line corresponds to a recipient mouse. The results of four mice in which leukemia spontaneously regressed (Exp. 3 in Table 1 ) are shown. (E) FACS analysis of BM from non-irradiated wild-type or Rag2 -/- mice transplanted with 3 × 10 4 of MLL/AF9 leukemia cells (OVA-). Mice were analyzed 3 weeks after transplant.
Figure Legend Snippet: Spontaneous regression of leukemia was observed in the presence, but not in the absence, of adaptive immunity. (A) Flow-cytometry analyses of BM cells of non-irradiated wild-type recipients 7 days after transplantation with different numbers (3 × 10 3 , 3 × 10 4 , or 3 × 10 5 ) of MLL/AF9-OVA leukemia cells. (B) FACS analysis of BM from non-irradiated wild-type or Rag2 -/- mice transplanted with 3 × 10 4 MLL/AF9-OVA leukemia cells. Mice were analyzed 3 weeks after transplant. (C) Kaplan–Meier curves for overall survival of non-irradiated wild-type (n = 7) or Rag2 -/- (n = 3) recipients transplanted with 3 × 10 4 MLL/AF9-OVA leukemia cells. (D) Percentages of GFP + leukemia cells in BM after transplantation into non-irradiated wild-type mice were examined every week. Each dot and line corresponds to a recipient mouse. The results of four mice in which leukemia spontaneously regressed (Exp. 3 in Table 1 ) are shown. (E) FACS analysis of BM from non-irradiated wild-type or Rag2 -/- mice transplanted with 3 × 10 4 of MLL/AF9 leukemia cells (OVA-). Mice were analyzed 3 weeks after transplant.

Techniques Used: Flow Cytometry, Cytometry, Irradiation, Transplantation Assay, FACS, Mouse Assay

Establishment of MLL/AF9 leukemia cells expressing a model tumor antigen. (A) Scheme for establishment of MLL/AF9-HPC-OVA cells. (B) FACS analysis of MLL/AF9-HPC-OVA cells. Blue lines represent MLL/AF9-HPC cells. (C) 51 Cr-release assay using activated CD8 + T cells from the OT-1 mouse as effector cells. MLL/AF9-HPC-OVA or MLL/AF9-HPC cells were used as targets. E:T ratio denotes effector-per-target ratio. (D) Kaplan–Meier curves for overall survival of wild-type mice that received 1 × 10 6 MLL/AF9-HPC-OVA cells in the third transplant (n = 5). Recipient mice were not irradiated. Results from the first and second transplants are shown in S1 Fig . (E) May–Giemsa staining of MLL/AF9-OVA leukemia cells that developed in non-irradiated recipients (Magnification: 400×). (F) Flow-cytometry analysis of GFP + BM cells from mice with leukemia. Blue lines represent MLL/AF9-HPC cells.
Figure Legend Snippet: Establishment of MLL/AF9 leukemia cells expressing a model tumor antigen. (A) Scheme for establishment of MLL/AF9-HPC-OVA cells. (B) FACS analysis of MLL/AF9-HPC-OVA cells. Blue lines represent MLL/AF9-HPC cells. (C) 51 Cr-release assay using activated CD8 + T cells from the OT-1 mouse as effector cells. MLL/AF9-HPC-OVA or MLL/AF9-HPC cells were used as targets. E:T ratio denotes effector-per-target ratio. (D) Kaplan–Meier curves for overall survival of wild-type mice that received 1 × 10 6 MLL/AF9-HPC-OVA cells in the third transplant (n = 5). Recipient mice were not irradiated. Results from the first and second transplants are shown in S1 Fig . (E) May–Giemsa staining of MLL/AF9-OVA leukemia cells that developed in non-irradiated recipients (Magnification: 400×). (F) Flow-cytometry analysis of GFP + BM cells from mice with leukemia. Blue lines represent MLL/AF9-HPC cells.

Techniques Used: Expressing, FACS, Release Assay, Mouse Assay, Irradiation, Staining, Flow Cytometry, Cytometry

37) Product Images from "Dynamic changes in the regulatory T-cell heterogeneity and function by murine IL-2 mutein"

Article Title: Dynamic changes in the regulatory T-cell heterogeneity and function by murine IL-2 mutein

Journal: Life Science Alliance

doi: 10.26508/lsa.201900520

Tnfrsf9 + Il1rl1 + Tregs are superior in suppression functional assay of Tregs in vitro. (A) Expression of Il1rl1 ( ST2 ) and Tnfrsf9 ( 4-1BB ) in RNA single-cell analysis of Tregs from the spleen ( top ) or lung ( bottom ) of isotype control–treated (Iso) or murine IL-2M–treated mice (IL-2M). (B) Expression of ST2 and 4-1BB using FACS analysis from the spleen ( top ) or lung ( bottom ) of Iso or IL-2M–treated mice. (C) Quantification of ST2+4-1BB+ cells in Foxp3+ Tregs. (B, C) Results are re presentative of two independent experiments, using three mice in each experiment. (D) Fopx3-EGFP+ Tregs were stained based on ST2 and 4-1BB expression. Single-cell suspension from two IL-2M–treated mice was combined and four quadrants of Fopx3-EGFP+ Tregs were sorted based on ST2 and 4-1BB. In vitro suppression assays were performed in duplicate using four populations of sorted Tregs or without Tregs. Experiments were repeated using three mice and in triplicate (data not shown). (E) Suppression function of four Treg sub-populations on IFNγ production. Four quadrants of Fopx3-EGFP+ Tregs were sorted based on ST2 and 4-1BB. Tregs were cocultured with effector T cells and APCs differentiated under Th1 skewing condition. IFNγ intracellular staining under the conditions using Tregs from PBS- or IL-2M–treated mice. No Tregs were added in one condition as a control. (B, C, D, E) Results are representative of two independent experiments, using 2–3 mice in each experiment. (C, D) Statistics: (C, D), one-way ANOVA for multiple comparisons. **0.001
Figure Legend Snippet: Tnfrsf9 + Il1rl1 + Tregs are superior in suppression functional assay of Tregs in vitro. (A) Expression of Il1rl1 ( ST2 ) and Tnfrsf9 ( 4-1BB ) in RNA single-cell analysis of Tregs from the spleen ( top ) or lung ( bottom ) of isotype control–treated (Iso) or murine IL-2M–treated mice (IL-2M). (B) Expression of ST2 and 4-1BB using FACS analysis from the spleen ( top ) or lung ( bottom ) of Iso or IL-2M–treated mice. (C) Quantification of ST2+4-1BB+ cells in Foxp3+ Tregs. (B, C) Results are re presentative of two independent experiments, using three mice in each experiment. (D) Fopx3-EGFP+ Tregs were stained based on ST2 and 4-1BB expression. Single-cell suspension from two IL-2M–treated mice was combined and four quadrants of Fopx3-EGFP+ Tregs were sorted based on ST2 and 4-1BB. In vitro suppression assays were performed in duplicate using four populations of sorted Tregs or without Tregs. Experiments were repeated using three mice and in triplicate (data not shown). (E) Suppression function of four Treg sub-populations on IFNγ production. Four quadrants of Fopx3-EGFP+ Tregs were sorted based on ST2 and 4-1BB. Tregs were cocultured with effector T cells and APCs differentiated under Th1 skewing condition. IFNγ intracellular staining under the conditions using Tregs from PBS- or IL-2M–treated mice. No Tregs were added in one condition as a control. (B, C, D, E) Results are representative of two independent experiments, using 2–3 mice in each experiment. (C, D) Statistics: (C, D), one-way ANOVA for multiple comparisons. **0.001

Techniques Used: Functional Assay, In Vitro, Expressing, Single-cell Analysis, Mouse Assay, FACS, Staining

38) Product Images from "Loss of the molecular clock in myeloid cells exacerbates T cell-mediated CNS autoimmune disease"

Article Title: Loss of the molecular clock in myeloid cells exacerbates T cell-mediated CNS autoimmune disease

Journal: Nature Communications

doi: 10.1038/s41467-017-02111-0

Severity of EAE is dependent upon time-of-day at point of immunization. a Lymph node (LN) cells isolated at either ZT6 or 18 were stimulated with medium or Mycobacterium tuberculosis (MTB) (100 µg/ml). Supernatants were tested by ELISA for IL-1β and TNF after 24 h. b Daily clinical score for myelin oligodendrocyte glycoprotein (MOG 35–55 ) + complete Freund’s adjuvant (CFA) + pertussis toxin (PT)-induced EAE in female C57BL/6 mice, immunized at either ZT6 or ZT18. Statistics by Kruskal–Wallis test on indicated days of 12 mice per group and Mann–Whitney test of area under the curve. c EAE clinical scores in Bmal1 Myeloid+/+ and Bmal1 Myeloid−/− female mice immunized with MOG + CFA and PT at either ZT6 or ZT18. Statistics by Kruskal–Wallis test on indicated days for 7–12 mice per group and Mann–Whitney test of area under the curve. d , e Peritoneal exudate cells were removed from 3 d MOG 35–55 + CFA + PT ZT6 or ZT18-immunized or naive female C57BL/6 mice ( n = 4–5). d FACS was performed directly ex vivo gating on CD11b + cells and examining Ly6C hi and Ly6G + populations ( n = 4–5). e Intracellular FACS for IL-1β production gating on CD11b + Ly6C hi or CD11b + Ly6G + populations ( n = 4–5). Statistics were performed by Mann–Whitney U test ( a , d , e ). All data is presented as means±standard error of the mean (SEM). * p
Figure Legend Snippet: Severity of EAE is dependent upon time-of-day at point of immunization. a Lymph node (LN) cells isolated at either ZT6 or 18 were stimulated with medium or Mycobacterium tuberculosis (MTB) (100 µg/ml). Supernatants were tested by ELISA for IL-1β and TNF after 24 h. b Daily clinical score for myelin oligodendrocyte glycoprotein (MOG 35–55 ) + complete Freund’s adjuvant (CFA) + pertussis toxin (PT)-induced EAE in female C57BL/6 mice, immunized at either ZT6 or ZT18. Statistics by Kruskal–Wallis test on indicated days of 12 mice per group and Mann–Whitney test of area under the curve. c EAE clinical scores in Bmal1 Myeloid+/+ and Bmal1 Myeloid−/− female mice immunized with MOG + CFA and PT at either ZT6 or ZT18. Statistics by Kruskal–Wallis test on indicated days for 7–12 mice per group and Mann–Whitney test of area under the curve. d , e Peritoneal exudate cells were removed from 3 d MOG 35–55 + CFA + PT ZT6 or ZT18-immunized or naive female C57BL/6 mice ( n = 4–5). d FACS was performed directly ex vivo gating on CD11b + cells and examining Ly6C hi and Ly6G + populations ( n = 4–5). e Intracellular FACS for IL-1β production gating on CD11b + Ly6C hi or CD11b + Ly6G + populations ( n = 4–5). Statistics were performed by Mann–Whitney U test ( a , d , e ). All data is presented as means±standard error of the mean (SEM). * p

Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay, Mouse Assay, MANN-WHITNEY, FACS, Ex Vivo

Enhanced accumulation of inflammatory CD11c + Ly6C + cells producing IL-1β in the CNS of Bmal1 Myeloid−/− mice during EAE. a – c Bmal1 Myeloid+/+ and Bmal1 Myeloid−/− mice were immunized to develop experimental autoimmune encephalomyelitis (EAE) with myelin oligodendrocyte glycoprotein (MOG 35–55 ) + complete Freund’s adjuvant (CFA) on d 0 and with pertussis toxin (PT) (125 ng/mouse) on d 0 and d 2 ( n = 6). a D 10 post EAE induction live cells infiltrating into the spinal cord were FACS stained for CD45 ( n = 6). b Live CD45 + cells were examined for Ly6C v CD11b or CCR2 v Ly6C ( n = 6). c IL-1β expression was determined in live CD45 + CD11b + Ly6C + cells ( n = 6). d 10 d post immunization CD11b and Ly6C populations infiltrating the spinal cord of Bmal1 Myeloid−/− mice were examined for IL-1β expression by FACS, gating on live CD45 + cells ( n = 6). Data as means ±standard error of the mean (SEM). Statistics were performed by Mann–Whitney U test a – d . * p
Figure Legend Snippet: Enhanced accumulation of inflammatory CD11c + Ly6C + cells producing IL-1β in the CNS of Bmal1 Myeloid−/− mice during EAE. a – c Bmal1 Myeloid+/+ and Bmal1 Myeloid−/− mice were immunized to develop experimental autoimmune encephalomyelitis (EAE) with myelin oligodendrocyte glycoprotein (MOG 35–55 ) + complete Freund’s adjuvant (CFA) on d 0 and with pertussis toxin (PT) (125 ng/mouse) on d 0 and d 2 ( n = 6). a D 10 post EAE induction live cells infiltrating into the spinal cord were FACS stained for CD45 ( n = 6). b Live CD45 + cells were examined for Ly6C v CD11b or CCR2 v Ly6C ( n = 6). c IL-1β expression was determined in live CD45 + CD11b + Ly6C + cells ( n = 6). d 10 d post immunization CD11b and Ly6C populations infiltrating the spinal cord of Bmal1 Myeloid−/− mice were examined for IL-1β expression by FACS, gating on live CD45 + cells ( n = 6). Data as means ±standard error of the mean (SEM). Statistics were performed by Mann–Whitney U test a – d . * p

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

39) Product Images from "A viral Sm-class RNA base-pairs with mRNAs and recruits miRNAs to inhibit apoptosis"

Article Title: A viral Sm-class RNA base-pairs with mRNAs and recruits miRNAs to inhibit apoptosis

Journal: Nature

doi: 10.1038/nature24034

HSUR 2 represses target mRNAs in the human B cell line BJAB BJAB cells were transiently transfected with plasmids pBS-GFP (Empty vector, white bars), pBS-GFP-HSUR2 (HSUR 2), or pBS-GFP-ΔHSUR2 (ΔHSUR2), FACS-sorted for GFP expression, and analyzed as described in ( t for full description of statistics). Fig. 2e
Figure Legend Snippet: HSUR 2 represses target mRNAs in the human B cell line BJAB BJAB cells were transiently transfected with plasmids pBS-GFP (Empty vector, white bars), pBS-GFP-HSUR2 (HSUR 2), or pBS-GFP-ΔHSUR2 (ΔHSUR2), FACS-sorted for GFP expression, and analyzed as described in ( t for full description of statistics). Fig. 2e

Techniques Used: Transfection, Plasmid Preparation, FACS, Expressing

HSURs 3, 4, 6, and 7 are not required for HSUR 2-mediated mRNA repression a, are not depicted. b, Plasmid constructs used for transient expression of HSUR 2. HSUR 2 expression signals (distal sequence element, DSE; proximal sequence element, PSE) are represented by magenta boxes, whereas the 3′-end processing signal (3′-box) is represented by a purple box. c, Northern blot analysis of GSML cells transiently transfected with pBS-GFP (lane 1), pBS-GFP-H1-5 (lane 2), pBS-GFP-H3-7 (lane 3), and both pBS-GFP-H1-5 and pBS-GFP-H3-7 (lane 4). U2 snRNA provides a loading control. d, GSML cells were transiently transfected with the plasmid constructs described in b , FACS-sorted for GFP expression, and analyzed as described in (two-sided t for full description of statistics). Fig. 2e
Figure Legend Snippet: HSURs 3, 4, 6, and 7 are not required for HSUR 2-mediated mRNA repression a, are not depicted. b, Plasmid constructs used for transient expression of HSUR 2. HSUR 2 expression signals (distal sequence element, DSE; proximal sequence element, PSE) are represented by magenta boxes, whereas the 3′-end processing signal (3′-box) is represented by a purple box. c, Northern blot analysis of GSML cells transiently transfected with pBS-GFP (lane 1), pBS-GFP-H1-5 (lane 2), pBS-GFP-H3-7 (lane 3), and both pBS-GFP-H1-5 and pBS-GFP-H3-7 (lane 4). U2 snRNA provides a loading control. d, GSML cells were transiently transfected with the plasmid constructs described in b , FACS-sorted for GFP expression, and analyzed as described in (two-sided t for full description of statistics). Fig. 2e

Techniques Used: Plasmid Preparation, Construct, Expressing, Sequencing, Northern Blot, Transfection, FACS

40) Product Images from "TNFα-Signaling Modulates the Kinase Activity of Human Effector Treg and Regulates IL-17A Expression"

Article Title: TNFα-Signaling Modulates the Kinase Activity of Human Effector Treg and Regulates IL-17A Expression

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.03047

Reciprocal TNFα and IL-17A expression in human naïve Treg and eff Treg cells. (A) An example of the FACS sorting strategy of naïve Treg and eff Treg based on CD4, CD45RA, and CD25 expression (I. dotplots), post-sorting analysis (II. dotplots) and confirmation of FOXP3 expression in the sorted cell population (III. histograms). Conventional CD4 + CD45RA − CD25 − naïve T cells (T naïve ), and CD4 + CD45RA − CD25 − memory T cells (T mem ) were sorted and displayed for comparison of FOXP3 expression levels (III). (B) RT-qPCR gene expression of TNFA, IL17A, IL17F , and RORC in naïve Treg and eff Treg after 20 h of PMA and ionomycin stimulation ( n = 12). (C) Heatmap displaying the fold change of transcripts expression in eff Treg within different donors (rows). naïve Treg were used as reference to calculate the fold change. (D) Multiple correlation matrix depicting the correlation of gene expression in both Treg subsets ( naïve Treg [open dots] and eff Treg [closed dots]). Sample distribution (histogram) is shown, linear regression is also plotted (red lines), whereas p -value significance and r- values are displayed based on Pearson correlation test. Y and X axes depict the log10-fold change of TNFA, IL17A, IL17F , and RORC expression. Each column represents a gene; in every intersection (rows), we observe the correlation between genes. (E) Presence of the cytokines TNFα and IL-17A in culture supernatant after overnight stimulation of Treg subsets using PMA and ionomycin. Cytokines were measured using Luminex ( n = 14). (F) Presence of TNFα and IL-17A in culture supernatants of αCD3/CD28/rhIL-2 activated Treg subsets after 5 days of culture ( n = 3, mean ± SEM). For statistical analysis, Wilcoxon matched-pairs signed-ranks test (B,E) , or two-way ANOVA followed by a Bonferroni post-hoc test (F) were used. * p
Figure Legend Snippet: Reciprocal TNFα and IL-17A expression in human naïve Treg and eff Treg cells. (A) An example of the FACS sorting strategy of naïve Treg and eff Treg based on CD4, CD45RA, and CD25 expression (I. dotplots), post-sorting analysis (II. dotplots) and confirmation of FOXP3 expression in the sorted cell population (III. histograms). Conventional CD4 + CD45RA − CD25 − naïve T cells (T naïve ), and CD4 + CD45RA − CD25 − memory T cells (T mem ) were sorted and displayed for comparison of FOXP3 expression levels (III). (B) RT-qPCR gene expression of TNFA, IL17A, IL17F , and RORC in naïve Treg and eff Treg after 20 h of PMA and ionomycin stimulation ( n = 12). (C) Heatmap displaying the fold change of transcripts expression in eff Treg within different donors (rows). naïve Treg were used as reference to calculate the fold change. (D) Multiple correlation matrix depicting the correlation of gene expression in both Treg subsets ( naïve Treg [open dots] and eff Treg [closed dots]). Sample distribution (histogram) is shown, linear regression is also plotted (red lines), whereas p -value significance and r- values are displayed based on Pearson correlation test. Y and X axes depict the log10-fold change of TNFA, IL17A, IL17F , and RORC expression. Each column represents a gene; in every intersection (rows), we observe the correlation between genes. (E) Presence of the cytokines TNFα and IL-17A in culture supernatant after overnight stimulation of Treg subsets using PMA and ionomycin. Cytokines were measured using Luminex ( n = 14). (F) Presence of TNFα and IL-17A in culture supernatants of αCD3/CD28/rhIL-2 activated Treg subsets after 5 days of culture ( n = 3, mean ± SEM). For statistical analysis, Wilcoxon matched-pairs signed-ranks test (B,E) , or two-way ANOVA followed by a Bonferroni post-hoc test (F) were used. * p

Techniques Used: Expressing, FACS, Quantitative RT-PCR, Luminex

Related Articles

FACS:

Article Title: B7-CD28 co-stimulation modulates central tolerance via thymic clonal deletion and Treg generation through distinct mechanisms
Article Snippet: For active-caspase3 staining, cells were fixed and permeabilized with BD Fix/Perm kit (BD Bioscience) and then stained with anti-active caspase3 or isotype control Ab for 30 min at RT. .. Data were collected with a FACS Calibur II, FACS LSR II, FACS Fortessa, or FACS Aria III (BD Biosciences) flow cytometer and analyzed with FlowJo software (Tree Star). .. Gating strategies for flow cytometry analysis are provided in Supplementary Fig. .

Article Title: Developmental pathway of CD4+CD8− medullary thymocytes during mouse ontogeny and its defect in Aire−/− mice
Article Snippet: For the isolation of different subsets of CD4SP medullary thymocytes, single-thymocyte suspension was treated with anti-CD8 (3.155) mAb and complement (guinea pig sera), and the removal of CD8+ cells was confirmed by staining with another anti-CD8 mAb (53–6.7). .. The resulting viable cells were then stained for CD4, TCRβ, CD69, and 6C10 or Qa-2 and sorted into various subsets with FACS Aria (BD Biosciences). ..

Article Title: Tolfenamic Acid Induces Apoptosis and Growth Inhibition in Head and Neck Cancer: Involvement of NAG-1 Expression
Article Snippet: .. Apoptosis was detected using a FACS Canto system (BD Biosciences, Bedford, MA), with excitation and emission settings of 488 and 530 nm, respectively. .. Mitochondrial membrane potential (MMP) assay MMP of intact cells was measured by flow cytometry with the lipophilic cationic probe 5,5 V,6,6 V-tetrachloro-1,1 V 3,3 V-tetra ethylbenzimidazolcarbocyanine iodide (JC-1; Molecular Probes, Eugene, OR).

Article Title: Benzene-Induced Aberrant miRNA Expression Profile in Hematopoietic Progenitor Cells in C57BL/6 Mice
Article Snippet: .. Sorting of HSPCs through Flow Cytometry and Total RNA Extraction Lin− and Lin− c-Kit+ cells were obtained with the FACS Aria™ II flow cytometer (Becton Dickinson, USA) to analyze different miRNA expression profiles between the control and benzene exposure groups. .. To obtain sufficient cells per group for Illumina sequencing and qRT-PCR analysis, we pooled Lin− cells from two mice per group and Lin− c-Kit+ cells from three mice per group.

Flow Cytometry:

Article Title: B7-CD28 co-stimulation modulates central tolerance via thymic clonal deletion and Treg generation through distinct mechanisms
Article Snippet: For active-caspase3 staining, cells were fixed and permeabilized with BD Fix/Perm kit (BD Bioscience) and then stained with anti-active caspase3 or isotype control Ab for 30 min at RT. .. Data were collected with a FACS Calibur II, FACS LSR II, FACS Fortessa, or FACS Aria III (BD Biosciences) flow cytometer and analyzed with FlowJo software (Tree Star). .. Gating strategies for flow cytometry analysis are provided in Supplementary Fig. .

Article Title: Elucidating the fundamental fibrotic processes driving abdominal adhesion formation
Article Snippet: Antibodies against the following cell surface markers primarily or secondarily conjugated to the same fluorophore were used for exclusion of “lineage” cells in mouse and human specimens in order to isolate fibroblasts in an unbiased manner: CD45, CD31, Ter119, Tie2, CD324, and CD326. .. For phospho-specific flow-cytometry analysis, a single-cell suspension was prepared using manual tissue dispersion rather than enzymatic digestion to preserve phosphorylated signal, and then prepared using the BD Biosciences Cytofix/Cytoperm™ kit according to manufacturer’s instructions. .. Phosphorylated protein analysis was conducted using the FACS Aria II system.

Article Title: Benzene-Induced Aberrant miRNA Expression Profile in Hematopoietic Progenitor Cells in C57BL/6 Mice
Article Snippet: .. Sorting of HSPCs through Flow Cytometry and Total RNA Extraction Lin− and Lin− c-Kit+ cells were obtained with the FACS Aria™ II flow cytometer (Becton Dickinson, USA) to analyze different miRNA expression profiles between the control and benzene exposure groups. .. To obtain sufficient cells per group for Illumina sequencing and qRT-PCR analysis, we pooled Lin− cells from two mice per group and Lin− c-Kit+ cells from three mice per group.

Software:

Article Title: B7-CD28 co-stimulation modulates central tolerance via thymic clonal deletion and Treg generation through distinct mechanisms
Article Snippet: For active-caspase3 staining, cells were fixed and permeabilized with BD Fix/Perm kit (BD Bioscience) and then stained with anti-active caspase3 or isotype control Ab for 30 min at RT. .. Data were collected with a FACS Calibur II, FACS LSR II, FACS Fortessa, or FACS Aria III (BD Biosciences) flow cytometer and analyzed with FlowJo software (Tree Star). .. Gating strategies for flow cytometry analysis are provided in Supplementary Fig. .

Fluorescence:

Article Title: Loss of SIMPL Compromises TNF? Dependent Survival of Hematopoietic Progenitors
Article Snippet: APC conjugated Annexin V and 7-amino-actinomycin were obtained from BD Biosciences (San Diego, CA). .. Apoptosis assays were performed as described by the manufacturers (BD Biosciences) and fluorescence was quantitated by FACSCalibur or the FACScan (BD Biosciences). .. For the whole bone marrow apoptosis assay of transplanted mice, CD45.2+ cells were sorted with PE-CD45.2 and PE IgG2A isotype by FACS eBioscience (San Diego, CA).

Mouse Assay:

Article Title: A population of innate myelolymphoblastoid effector cell expanded by inactivation of mTOR complex 1 in mice
Article Snippet: The enrichment of Lin- BM cells was performed using MACS beads from mouse Lineage Cell Depletion Kit (Miltenyi Biotec). .. CD11b+ Gr-1- BM cells from Rptor F/F , Mx1-Cre mice, Lin- CD11b+ PD-L1+ IMLECs / LSK/CLP/ CMP populations from Raptor Ctrl/cKO or WT mice, CD11b+ Gr-1+ / CD11b- Gr-1- BM populations from Rptor F/F , Lyz2-Cre+/+ mice, CD11bhigh Gr-1- F4/80+ peritoneal macrophages were sorted using FACSAria II or Influx cell sorter (BD Biosciences). .. BM cells transplantation C57BL/6 Ly5.2 (CD45.1+ ) recipient mice at the age of 6–12 weeks old were lethally irradiated for total 900–1,100 rads with a Cs-137 γ-ray source or a RS 2000 X-ray irradiator (Rad Source Technologies, Inc.).

Staining:

Article Title: Developmental pathway of CD4+CD8− medullary thymocytes during mouse ontogeny and its defect in Aire−/− mice
Article Snippet: For the isolation of different subsets of CD4SP medullary thymocytes, single-thymocyte suspension was treated with anti-CD8 (3.155) mAb and complement (guinea pig sera), and the removal of CD8+ cells was confirmed by staining with another anti-CD8 mAb (53–6.7). .. The resulting viable cells were then stained for CD4, TCRβ, CD69, and 6C10 or Qa-2 and sorted into various subsets with FACS Aria (BD Biosciences). ..

Cytometry:

Article Title: Benzene-Induced Aberrant miRNA Expression Profile in Hematopoietic Progenitor Cells in C57BL/6 Mice
Article Snippet: .. Sorting of HSPCs through Flow Cytometry and Total RNA Extraction Lin− and Lin− c-Kit+ cells were obtained with the FACS Aria™ II flow cytometer (Becton Dickinson, USA) to analyze different miRNA expression profiles between the control and benzene exposure groups. .. To obtain sufficient cells per group for Illumina sequencing and qRT-PCR analysis, we pooled Lin− cells from two mice per group and Lin− c-Kit+ cells from three mice per group.

RNA Extraction:

Article Title: Benzene-Induced Aberrant miRNA Expression Profile in Hematopoietic Progenitor Cells in C57BL/6 Mice
Article Snippet: .. Sorting of HSPCs through Flow Cytometry and Total RNA Extraction Lin− and Lin− c-Kit+ cells were obtained with the FACS Aria™ II flow cytometer (Becton Dickinson, USA) to analyze different miRNA expression profiles between the control and benzene exposure groups. .. To obtain sufficient cells per group for Illumina sequencing and qRT-PCR analysis, we pooled Lin− cells from two mice per group and Lin− c-Kit+ cells from three mice per group.

Expressing:

Article Title: Benzene-Induced Aberrant miRNA Expression Profile in Hematopoietic Progenitor Cells in C57BL/6 Mice
Article Snippet: .. Sorting of HSPCs through Flow Cytometry and Total RNA Extraction Lin− and Lin− c-Kit+ cells were obtained with the FACS Aria™ II flow cytometer (Becton Dickinson, USA) to analyze different miRNA expression profiles between the control and benzene exposure groups. .. To obtain sufficient cells per group for Illumina sequencing and qRT-PCR analysis, we pooled Lin− cells from two mice per group and Lin− c-Kit+ cells from three mice per group.

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    Becton Dickinson facs sorted
    <t>FACS</t> analysis and immunohistochemistry of xenograft <t>ALDH</t> + 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
    Facs Sorted, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson fluorescence activated cell sorting facs analysis
    IL‐6 treatment induced prostasphere formation in IL‐6 nonexpressing PCa cell line cultures. (A) Effect of IL‐6 treatment relative to vehicle control on the numbers of prostaspheres in 7‐day cultures of MDA‐PCa‐2b cells. (B) Effect of IL‐6 treatment (72‐h) on viability of MDA‐PCa‐2b cells, run in triplicate and repeated twice. (C) Effect of IL‐6 treatment relative to vehicle control on the numbers of prostaspheres in 7‐day cultures of RC77T cells. (D) Effect of IL‐6 on viability of RC77T cells, 7‐day treatment run in triplicate and repeated twice. (E) Effect of 7‐day IL‐6 treatment on the numbers of PC3 prostaspheres. (F) Effect of IL‐6 receptor inhibitor tocilizumab (10 μ m , 7 days) on prostaspheres in PC3 cultures. (G) Impact of IL‐6 treatment on the percentage of <t>CSCs</t> in other cell lines in our panel measured by <t>FACS</t> analysis. Cells were treated with IL‐6 at 10 ng·mL −1 for 7 or 14 days for RC77T. The fraction of CSCs relative to total cell count was measured based on CSC triple‐marker‐positive status (CD44+/CD133+/EpCAM+). The results are presented as fold‐change, IL‐6 treated vs. control. Prostasphere assay and FACS data are representative of repeated experiments and are the average of three independent biological replicates. * P ≤ 0.05, ** P ≤ 0.01.
    Fluorescence Activated Cell Sorting Facs Analysis, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The development of iNKT1 (Plzf low T-bet high ), iNKT2 (Plzf high T-bet lowh ) and iNKT17 (Rorγt high ) cells in Gfi1 -deficient mice. <t>iNKT</t> cells were purified by <t>FACS</t> sorting and the intracellular staining of the indicated transcription factors was performed. The expression of Plzf and T-bet in the thymus (A) and the indicated peripheral organs (C) , and the patterns of Plzf and Rorγt in the thymus (B) and the indicated peripheral organs (D) are shown.
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    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

    Journal: BMC Cancer

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

    doi: 10.1186/s12885-016-2466-z

    Figure Lengend 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

    Article Snippet: To induce subcutaneous human colon cancer xenografts, FACS-sorted 1 × 104 ALDH+ HCT-116 cells were suspended in a 1:1 mixture of PBS:Matrigel (BD Biosciences), and 100 μl of the mixture was injected subcutaneously into each of both flanks of the mice.

    Techniques: 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

    Journal: BMC Cancer

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

    doi: 10.1186/s12885-016-2466-z

    Figure Lengend 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

    Article Snippet: To induce subcutaneous human colon cancer xenografts, FACS-sorted 1 × 104 ALDH+ HCT-116 cells were suspended in a 1:1 mixture of PBS:Matrigel (BD Biosciences), and 100 μl of the mixture was injected subcutaneously into each of both flanks of the mice.

    Techniques: FACS, Mouse Assay

    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

    IL‐6 treatment induced prostasphere formation in IL‐6 nonexpressing PCa cell line cultures. (A) Effect of IL‐6 treatment relative to vehicle control on the numbers of prostaspheres in 7‐day cultures of MDA‐PCa‐2b cells. (B) Effect of IL‐6 treatment (72‐h) on viability of MDA‐PCa‐2b cells, run in triplicate and repeated twice. (C) Effect of IL‐6 treatment relative to vehicle control on the numbers of prostaspheres in 7‐day cultures of RC77T cells. (D) Effect of IL‐6 on viability of RC77T cells, 7‐day treatment run in triplicate and repeated twice. (E) Effect of 7‐day IL‐6 treatment on the numbers of PC3 prostaspheres. (F) Effect of IL‐6 receptor inhibitor tocilizumab (10 μ m , 7 days) on prostaspheres in PC3 cultures. (G) Impact of IL‐6 treatment on the percentage of CSCs in other cell lines in our panel measured by FACS analysis. Cells were treated with IL‐6 at 10 ng·mL −1 for 7 or 14 days for RC77T. The fraction of CSCs relative to total cell count was measured based on CSC triple‐marker‐positive status (CD44+/CD133+/EpCAM+). The results are presented as fold‐change, IL‐6 treated vs. control. Prostasphere assay and FACS data are representative of repeated experiments and are the average of three independent biological replicates. * P ≤ 0.05, ** P ≤ 0.01.

    Journal: Molecular Oncology

    Article Title: Exogenous IL‐6 induces mRNA splice variant MBD2_v2 to promote stemness in TP53 wild‐type, African American PCa cells

    doi: 10.1002/1878-0261.12316

    Figure Lengend Snippet: IL‐6 treatment induced prostasphere formation in IL‐6 nonexpressing PCa cell line cultures. (A) Effect of IL‐6 treatment relative to vehicle control on the numbers of prostaspheres in 7‐day cultures of MDA‐PCa‐2b cells. (B) Effect of IL‐6 treatment (72‐h) on viability of MDA‐PCa‐2b cells, run in triplicate and repeated twice. (C) Effect of IL‐6 treatment relative to vehicle control on the numbers of prostaspheres in 7‐day cultures of RC77T cells. (D) Effect of IL‐6 on viability of RC77T cells, 7‐day treatment run in triplicate and repeated twice. (E) Effect of 7‐day IL‐6 treatment on the numbers of PC3 prostaspheres. (F) Effect of IL‐6 receptor inhibitor tocilizumab (10 μ m , 7 days) on prostaspheres in PC3 cultures. (G) Impact of IL‐6 treatment on the percentage of CSCs in other cell lines in our panel measured by FACS analysis. Cells were treated with IL‐6 at 10 ng·mL −1 for 7 or 14 days for RC77T. The fraction of CSCs relative to total cell count was measured based on CSC triple‐marker‐positive status (CD44+/CD133+/EpCAM+). The results are presented as fold‐change, IL‐6 treated vs. control. Prostasphere assay and FACS data are representative of repeated experiments and are the average of three independent biological replicates. * P ≤ 0.05, ** P ≤ 0.01.

    Article Snippet: 2.5 Fluorescence‐activated cell sorting (FACS) analysis CSCs and total PCa cells were counted by fluorescence‐activated cell sorting (FACS) analysis, using the BD LSR II (BD Biosciences, San Jose, CA, USA), at the Karmanos Cancer Institute Microscopy, Imaging and Cytometry Resources Core.

    Techniques: Multiple Displacement Amplification, FACS, Cell Counting, Marker

    The development of iNKT1 (Plzf low T-bet high ), iNKT2 (Plzf high T-bet lowh ) and iNKT17 (Rorγt high ) cells in Gfi1 -deficient mice. iNKT cells were purified by FACS sorting and the intracellular staining of the indicated transcription factors was performed. The expression of Plzf and T-bet in the thymus (A) and the indicated peripheral organs (C) , and the patterns of Plzf and Rorγt in the thymus (B) and the indicated peripheral organs (D) are shown.

    Journal: PLoS ONE

    Article Title: The Transcriptional Repressor Gfi1 Plays a Critical Role in the Development of NKT1- and NKT2-Type iNKT Cells

    doi: 10.1371/journal.pone.0157395

    Figure Lengend Snippet: The development of iNKT1 (Plzf low T-bet high ), iNKT2 (Plzf high T-bet lowh ) and iNKT17 (Rorγt high ) cells in Gfi1 -deficient mice. iNKT cells were purified by FACS sorting and the intracellular staining of the indicated transcription factors was performed. The expression of Plzf and T-bet in the thymus (A) and the indicated peripheral organs (C) , and the patterns of Plzf and Rorγt in the thymus (B) and the indicated peripheral organs (D) are shown.

    Article Snippet: Isolation of iNKT cells by FACS sorting The iNKT cells were purified by FACS sorting using a FACS Aria (BD Biosciences).

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

    Reduction of IFN-γand Th2 cytokine production in Gfi1 -deficient iNKT cells. The results of the quantitative RT-PCR analysis of cytokine mRNA in iNKT cells from the spleen (A) , liver (B) and lung (C) of WT and Gfi1 -deficient mice. The iNKT cells were purified by FACS sorting and stimulated with PMA plus ionomycin for 2h. The results are presented relative to the mRNA expression of 18s ribosomal RNA with the standard deviation (n = 3). NS; not significant, *P

    Journal: PLoS ONE

    Article Title: The Transcriptional Repressor Gfi1 Plays a Critical Role in the Development of NKT1- and NKT2-Type iNKT Cells

    doi: 10.1371/journal.pone.0157395

    Figure Lengend Snippet: Reduction of IFN-γand Th2 cytokine production in Gfi1 -deficient iNKT cells. The results of the quantitative RT-PCR analysis of cytokine mRNA in iNKT cells from the spleen (A) , liver (B) and lung (C) of WT and Gfi1 -deficient mice. The iNKT cells were purified by FACS sorting and stimulated with PMA plus ionomycin for 2h. The results are presented relative to the mRNA expression of 18s ribosomal RNA with the standard deviation (n = 3). NS; not significant, *P

    Article Snippet: Isolation of iNKT cells by FACS sorting The iNKT cells were purified by FACS sorting using a FACS Aria (BD Biosciences).

    Techniques: Quantitative RT-PCR, Mouse Assay, Purification, FACS, Expressing, Standard Deviation

    The expression profile of transcriptional regulators in Gfi1 -deficient iNKT cells. The results of the quantitative RT-PCR analysis of transcriptional regulators in WT and Gfi1 -deficient iNKT cells from the thymus (A) , spleen (B) , liver (C) and lung (D) . Each population was purified by FACS sorting. The results are presented relative to the mRNA expression of 18s ribosomal RNA with the standard deviation (n = 3). (E) The results of the intracellular FACS analysis of Eomes, Gata3, Plzf, Rorγt and T-bet in iNKT cells from the indicated organs of WT and Gfi1 -deficient mice. Three independent experiments were performed with similar results. NS; not significant, *P

    Journal: PLoS ONE

    Article Title: The Transcriptional Repressor Gfi1 Plays a Critical Role in the Development of NKT1- and NKT2-Type iNKT Cells

    doi: 10.1371/journal.pone.0157395

    Figure Lengend Snippet: The expression profile of transcriptional regulators in Gfi1 -deficient iNKT cells. The results of the quantitative RT-PCR analysis of transcriptional regulators in WT and Gfi1 -deficient iNKT cells from the thymus (A) , spleen (B) , liver (C) and lung (D) . Each population was purified by FACS sorting. The results are presented relative to the mRNA expression of 18s ribosomal RNA with the standard deviation (n = 3). (E) The results of the intracellular FACS analysis of Eomes, Gata3, Plzf, Rorγt and T-bet in iNKT cells from the indicated organs of WT and Gfi1 -deficient mice. Three independent experiments were performed with similar results. NS; not significant, *P

    Article Snippet: Isolation of iNKT cells by FACS sorting The iNKT cells were purified by FACS sorting using a FACS Aria (BD Biosciences).

    Techniques: Expressing, Quantitative RT-PCR, Purification, FACS, Standard Deviation, Mouse Assay