hamster anti mouse cd69 pe  (Bio-Rad)

Journal: bioRxiv

Article Title: Epigenetic control of CD8 + T cell tissue homing and tissue resident memory T cell precursors by the histone methyltransferase SUV39H1

doi: 10.1101/2024.05.13.593994

Figure Lengend Snippet: A. Representative dot plots and quantification of CD44 and CD62L expression in CD8 + T cells from the indicated non lymphoid organs in conditional SUV39H1 KO (Cre+) or WT (Cre-) littermates. B. t-SNE plots of merged flow cytometry data gated on total CD8 + T cells from the liver of 5 conditional SUV39H1 KO (Cre+) and 5 WT littermates (Cre-) at steady state. See Supp Table 1. C. Representative dot plots and quantification of CD69 + CD103 - and CD49d + Ly6c - populations in CD44 + CD8 + T cells from liver of conditional SUV39H1 KO mice (Cre+) or WT littermates (Cre-) at steady state. D. Representative dot plots and quantification of intravascular staining negative (iv-) cells among lung CD8 + T cells and CD69 + CD103 - or CD49d + Ly6c - populations in iv-CD44 + CD8 + T cells from conditional SUV39H1 KO mice (Cre+) or WT littermates (Cre-) at steady state. Each dot represents a mouse, different shapes represent different experiments. * p<0.05 ; ** p<0.01 ; *** p< 0.001 ; **** p<0.0001 ; ns: not significant by multiple unpaired t test with Holm-Šídák correction ( A and B ) or Mann Whitney test ( C and D ).

Article Snippet: Cell suspensions were prepared in PBS and stained with Near IR (Fisher Scientific L34975) or Acqua LIVE/DEAD™ Fixable Cell Stain Kit (Fisher Scientific L34957) according to manufacturer’s instructions to exclude dead cells and then labelled in FACS buffer with the following antibodies: V500 Rat anti-Mouse CD4 (RM4-5, BD), eF450 CD5 Monoclonal Antibody (53-7.3, eBiosciences), FITC anti-CD8α (53-6.7, BD), PerCP-Cy5.5 CD8 α (53-6.7, BD), PE CD8 α Monoclonal Antibody (53-6.7, Ficher Scientific), BUV395 Rat Anti-Mouse CD8 β (H35-17.2, BD), APC anti-mouse CD25 Antibody (PC61, Biolegend), PerCP-Cy5.5 Rat Anti-Mouse CD44 (IM7, BD), APC Rat Anti-Mouse CD44 (IM7, BD), APC-R700 Rat Anti-Mouse CD44 (IM7, BD), eFluor 450 CD45.1 Monoclonal Antibody (A20, Fisher Scientific), PE Mouse Anti-Mouse CD45.1 (A20, BD), APC-Cy7 CD45.1 (A20, BD), PE Mouse Anti-Mouse CD45.2 (104, BD), APC Mouse anti-Mouse CD45.2 (104, BD), BV605 Hamster Anti-Rat/Mouse CD49a (Ha31/8, BD), BV786 Rat Anti-Mouse CD49d (R1-2, BD), PE/Cyanine7 anti-mouse CD49d Antibody (R1-2, Biolegend), PE-Cy7 Rat Anti-Mouse CD62L (MEL-14, BD), APC Rat Anti-Mouse CD62L (MEL-14, BD), APC/Cyanine7 anti-mouse CD62L Antibody (MEL-14, Biolegend), PerCP-Cy5.5 Hamster Anti-Mouse CD69 (H1.2F3, BD), PE-Cy7 Hamster Anti-Mouse CD69 (H1.2F3, BD), Brilliant Violet 605™ anti-mouse CD73 Antibody (TY/11.8, Biolegend), eFluor 450 CD103 Monoclonal Antibody (2E7, Fisher Scientific), eF660 CD127 (A7R34, eBiosciences), Brilliant Violet 711 anti-mouse CX3CR1 (SA011F11, Biolegend), BV711 CXCR3 (CXCR3-173, BD), Brilliant Violet 650™ anti-mouse Ly-6C Antibody (HK1.4, Biolegend), FITC anti-mouse Ly-6C (HK1.4, Biolegend), APC Ly-6C Monoclonal Antibody (HK1.4, eBiosciences), PE-CF594 Hamster Anti-Mouse KLRG1 (2F1, BD), BUV737 Hamster Anti-Mouse TCR β Chain (H57-597, BD), Alexa-Fluor®488 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-Cy™7 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-CF594 TCR β Chain (H57-597, BD), eFluor 450 TRCR V alpha 2 (B20.1, Invitrogen), PerCP/Cy5.5 anti-mouse TCR Vα2 (B20.1, Biolegend).

Techniques: Expressing, Flow Cytometry, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Epigenetic control of CD8 + T cell tissue homing and tissue resident memory T cell precursors by the histone methyltransferase SUV39H1

doi: 10.1101/2024.05.13.593994

Figure Lengend Snippet: A. Scheme of the experiment. B. Representative dot plots of WT (CD45.1-) and SUV39H1 KO (CD45.1+) OTI cells (Vα2+) in the indicated organs 33 days after adoptive transfer. C. Quantification of the ratio between SUV39H1 KO and WT OTI cells within each organ. D. Representative dot plot of intravascular (iv) staining ( left ) and expression of CD49d and Ly6c ( right ) in each population at day 34 post transfer in CD8 + T cells from lungs from Exp. 2 in C . E. Quantification of the percentage and absolute numbers of CD49d + Ly6c - cells among SUV39H1KO or WT OTI cells in the indicated organs from Exp 1 in C . Numbers represent the fold increase between KO and WT condition for each organ. F. Histogram of CD69 expression in iv-CD49d + Ly6c - CD8 + T cells and proportions of CD69+ cells among iv+ and iv-CD49d + Ly6c - CD8 + T cells in lungs from Exp. 4 in C . Numbers represent p values. Each dot represents a mouse, different shapes represent different experiments. * p<0.05 ; ** p<0.01 ; *** p< 0.001 ; **** p<0.0001 ; ns: not significant by one sample t test compared to 1 ( C ), multiple ratio paired t test with Holm-Šídák correction ( E ) and multiple paired t test ( F ).

Article Snippet: Cell suspensions were prepared in PBS and stained with Near IR (Fisher Scientific L34975) or Acqua LIVE/DEAD™ Fixable Cell Stain Kit (Fisher Scientific L34957) according to manufacturer’s instructions to exclude dead cells and then labelled in FACS buffer with the following antibodies: V500 Rat anti-Mouse CD4 (RM4-5, BD), eF450 CD5 Monoclonal Antibody (53-7.3, eBiosciences), FITC anti-CD8α (53-6.7, BD), PerCP-Cy5.5 CD8 α (53-6.7, BD), PE CD8 α Monoclonal Antibody (53-6.7, Ficher Scientific), BUV395 Rat Anti-Mouse CD8 β (H35-17.2, BD), APC anti-mouse CD25 Antibody (PC61, Biolegend), PerCP-Cy5.5 Rat Anti-Mouse CD44 (IM7, BD), APC Rat Anti-Mouse CD44 (IM7, BD), APC-R700 Rat Anti-Mouse CD44 (IM7, BD), eFluor 450 CD45.1 Monoclonal Antibody (A20, Fisher Scientific), PE Mouse Anti-Mouse CD45.1 (A20, BD), APC-Cy7 CD45.1 (A20, BD), PE Mouse Anti-Mouse CD45.2 (104, BD), APC Mouse anti-Mouse CD45.2 (104, BD), BV605 Hamster Anti-Rat/Mouse CD49a (Ha31/8, BD), BV786 Rat Anti-Mouse CD49d (R1-2, BD), PE/Cyanine7 anti-mouse CD49d Antibody (R1-2, Biolegend), PE-Cy7 Rat Anti-Mouse CD62L (MEL-14, BD), APC Rat Anti-Mouse CD62L (MEL-14, BD), APC/Cyanine7 anti-mouse CD62L Antibody (MEL-14, Biolegend), PerCP-Cy5.5 Hamster Anti-Mouse CD69 (H1.2F3, BD), PE-Cy7 Hamster Anti-Mouse CD69 (H1.2F3, BD), Brilliant Violet 605™ anti-mouse CD73 Antibody (TY/11.8, Biolegend), eFluor 450 CD103 Monoclonal Antibody (2E7, Fisher Scientific), eF660 CD127 (A7R34, eBiosciences), Brilliant Violet 711 anti-mouse CX3CR1 (SA011F11, Biolegend), BV711 CXCR3 (CXCR3-173, BD), Brilliant Violet 650™ anti-mouse Ly-6C Antibody (HK1.4, Biolegend), FITC anti-mouse Ly-6C (HK1.4, Biolegend), APC Ly-6C Monoclonal Antibody (HK1.4, eBiosciences), PE-CF594 Hamster Anti-Mouse KLRG1 (2F1, BD), BUV737 Hamster Anti-Mouse TCR β Chain (H57-597, BD), Alexa-Fluor®488 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-Cy™7 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-CF594 TCR β Chain (H57-597, BD), eFluor 450 TRCR V alpha 2 (B20.1, Invitrogen), PerCP/Cy5.5 anti-mouse TCR Vα2 (B20.1, Biolegend).

Techniques: Adoptive Transfer Assay, Staining, Expressing

Journal: bioRxiv

Article Title: Epigenetic control of CD8 + T cell tissue homing and tissue resident memory T cell precursors by the histone methyltransferase SUV39H1

doi: 10.1101/2024.05.13.593994

Figure Lengend Snippet: A. Scheme of the experiment. B. Representative dot plots and quantification of the normalized numbers of iv-CD44 + CD8 + T cells and the normalized percentages of CD49d + Ly6c - cells among iv-CD44 + CD8 + T cells in lungs 28 days post X31 infection ( Memory ). Values were normalized to the mean of X31-infected WT group for each experiment and then pooled. C. Representative dot plots and quantification of the number of iv-CD44 + CD8 + T cells in lungs 3 days post PR8 infection ( Recall ). D. Representative dot plots and quantification of the number of CD49d + Ly6c - and CD69 + CD103 - iv-CD44 + CD8 + T cells in lungs 3 days post PR8 infection ( Recall ). * p<0.05 ; ** p<0.01 ; *** p< 0.001 ; ns: not significant by ANOVA test with Šídák’s multiple comparisons test.

Article Snippet: Cell suspensions were prepared in PBS and stained with Near IR (Fisher Scientific L34975) or Acqua LIVE/DEAD™ Fixable Cell Stain Kit (Fisher Scientific L34957) according to manufacturer’s instructions to exclude dead cells and then labelled in FACS buffer with the following antibodies: V500 Rat anti-Mouse CD4 (RM4-5, BD), eF450 CD5 Monoclonal Antibody (53-7.3, eBiosciences), FITC anti-CD8α (53-6.7, BD), PerCP-Cy5.5 CD8 α (53-6.7, BD), PE CD8 α Monoclonal Antibody (53-6.7, Ficher Scientific), BUV395 Rat Anti-Mouse CD8 β (H35-17.2, BD), APC anti-mouse CD25 Antibody (PC61, Biolegend), PerCP-Cy5.5 Rat Anti-Mouse CD44 (IM7, BD), APC Rat Anti-Mouse CD44 (IM7, BD), APC-R700 Rat Anti-Mouse CD44 (IM7, BD), eFluor 450 CD45.1 Monoclonal Antibody (A20, Fisher Scientific), PE Mouse Anti-Mouse CD45.1 (A20, BD), APC-Cy7 CD45.1 (A20, BD), PE Mouse Anti-Mouse CD45.2 (104, BD), APC Mouse anti-Mouse CD45.2 (104, BD), BV605 Hamster Anti-Rat/Mouse CD49a (Ha31/8, BD), BV786 Rat Anti-Mouse CD49d (R1-2, BD), PE/Cyanine7 anti-mouse CD49d Antibody (R1-2, Biolegend), PE-Cy7 Rat Anti-Mouse CD62L (MEL-14, BD), APC Rat Anti-Mouse CD62L (MEL-14, BD), APC/Cyanine7 anti-mouse CD62L Antibody (MEL-14, Biolegend), PerCP-Cy5.5 Hamster Anti-Mouse CD69 (H1.2F3, BD), PE-Cy7 Hamster Anti-Mouse CD69 (H1.2F3, BD), Brilliant Violet 605™ anti-mouse CD73 Antibody (TY/11.8, Biolegend), eFluor 450 CD103 Monoclonal Antibody (2E7, Fisher Scientific), eF660 CD127 (A7R34, eBiosciences), Brilliant Violet 711 anti-mouse CX3CR1 (SA011F11, Biolegend), BV711 CXCR3 (CXCR3-173, BD), Brilliant Violet 650™ anti-mouse Ly-6C Antibody (HK1.4, Biolegend), FITC anti-mouse Ly-6C (HK1.4, Biolegend), APC Ly-6C Monoclonal Antibody (HK1.4, eBiosciences), PE-CF594 Hamster Anti-Mouse KLRG1 (2F1, BD), BUV737 Hamster Anti-Mouse TCR β Chain (H57-597, BD), Alexa-Fluor®488 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-Cy™7 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-CF594 TCR β Chain (H57-597, BD), eFluor 450 TRCR V alpha 2 (B20.1, Invitrogen), PerCP/Cy5.5 anti-mouse TCR Vα2 (B20.1, Biolegend).

Techniques: Infection

Journal: bioRxiv

Article Title: Epigenetic control of CD8 + T cell tissue homing and tissue resident memory T cell precursors by the histone methyltransferase SUV39H1

doi: 10.1101/2024.05.13.593994

Figure Lengend Snippet: A. Scheme of the experiment. B. Representative dot plots and quantification of the expression of CD69 and CD103 in SUV39H1-deficient and WT OTI cells 30 days post X31-OVA infection in lungs ( Memory ). C. Representative dot plots from lungs and quantification of the percentage of CD49d + Ly6c - cells among SUV39H1-deficient and WT OTI cells in the indicated organs and the percentage of CD69 + KLRG1 - or CD49a + CX3CR1 - cells among CD49d + Ly6c - iv-CD44 + OTI cells in lungs 9 days post X31-OVA infection ( Acute ). D. Scheme of the experiment. E. Gating strategy, representative dot plots and quantification of the percentage and numbers of WT and SUV39H1 KO OTI cells among CD69 + CD103 - and CD69 + CD103 + iv-CD44 + OTI cells in lungs 28 days post X31-OVA infection ( Memory ) and 27 days post adoptive transfer. p<0.05 ; ** p<0.01 ; *** p< 0.001 ; **** p<0.0001 ; ns: not significant by multiple ratio paired t test (B), (C) top and (E) and ratio pared t test (C) bottom. B, C and E : Representative data from 2 independent experiments.

Article Snippet: Cell suspensions were prepared in PBS and stained with Near IR (Fisher Scientific L34975) or Acqua LIVE/DEAD™ Fixable Cell Stain Kit (Fisher Scientific L34957) according to manufacturer’s instructions to exclude dead cells and then labelled in FACS buffer with the following antibodies: V500 Rat anti-Mouse CD4 (RM4-5, BD), eF450 CD5 Monoclonal Antibody (53-7.3, eBiosciences), FITC anti-CD8α (53-6.7, BD), PerCP-Cy5.5 CD8 α (53-6.7, BD), PE CD8 α Monoclonal Antibody (53-6.7, Ficher Scientific), BUV395 Rat Anti-Mouse CD8 β (H35-17.2, BD), APC anti-mouse CD25 Antibody (PC61, Biolegend), PerCP-Cy5.5 Rat Anti-Mouse CD44 (IM7, BD), APC Rat Anti-Mouse CD44 (IM7, BD), APC-R700 Rat Anti-Mouse CD44 (IM7, BD), eFluor 450 CD45.1 Monoclonal Antibody (A20, Fisher Scientific), PE Mouse Anti-Mouse CD45.1 (A20, BD), APC-Cy7 CD45.1 (A20, BD), PE Mouse Anti-Mouse CD45.2 (104, BD), APC Mouse anti-Mouse CD45.2 (104, BD), BV605 Hamster Anti-Rat/Mouse CD49a (Ha31/8, BD), BV786 Rat Anti-Mouse CD49d (R1-2, BD), PE/Cyanine7 anti-mouse CD49d Antibody (R1-2, Biolegend), PE-Cy7 Rat Anti-Mouse CD62L (MEL-14, BD), APC Rat Anti-Mouse CD62L (MEL-14, BD), APC/Cyanine7 anti-mouse CD62L Antibody (MEL-14, Biolegend), PerCP-Cy5.5 Hamster Anti-Mouse CD69 (H1.2F3, BD), PE-Cy7 Hamster Anti-Mouse CD69 (H1.2F3, BD), Brilliant Violet 605™ anti-mouse CD73 Antibody (TY/11.8, Biolegend), eFluor 450 CD103 Monoclonal Antibody (2E7, Fisher Scientific), eF660 CD127 (A7R34, eBiosciences), Brilliant Violet 711 anti-mouse CX3CR1 (SA011F11, Biolegend), BV711 CXCR3 (CXCR3-173, BD), Brilliant Violet 650™ anti-mouse Ly-6C Antibody (HK1.4, Biolegend), FITC anti-mouse Ly-6C (HK1.4, Biolegend), APC Ly-6C Monoclonal Antibody (HK1.4, eBiosciences), PE-CF594 Hamster Anti-Mouse KLRG1 (2F1, BD), BUV737 Hamster Anti-Mouse TCR β Chain (H57-597, BD), Alexa-Fluor®488 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-Cy™7 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-CF594 TCR β Chain (H57-597, BD), eFluor 450 TRCR V alpha 2 (B20.1, Invitrogen), PerCP/Cy5.5 anti-mouse TCR Vα2 (B20.1, Biolegend).

Techniques: Expressing, Infection, Adoptive Transfer Assay

Journal: Frontiers in Immunology

Article Title: CD5 blockade, a novel immune checkpoint inhibitor, enhances T cell anti-tumour immunity and delays tumour growth in mice harbouring poorly immunogenic 4T1 breast tumour homografts

doi: 10.3389/fimmu.2024.1256766

Figure Lengend Snippet: Gating strategy. Cells were gated side scatter area vs forward scatter area then Zombie Aqua dye (which penetrates non-viable cells but not viable cells; Biolegend, San Diego, USA) was used to gate on live cells only. After that, cells were gated on side scatter height vs side scatter area to exclude duplicate cells. Cells were then gated on CD3 marker and then on CD4 and CD8 markers. Lastly, cells were gated based on marker of interest (CD69, FasR, CD137 and CD107a) as shown on the following figures. The arrows indicate stepwise progression through each of the gating steps.

Article Snippet: To assess the levels of CD69, CD107a, CD137 and FasR on T cells by flow cytometry, lymphocytes obtained as described above were stained with the following antibodies: Brilliant Violet 711™ anti-mouse CD3 (BioLegend, San Diego, CA), Alexa Fluor ® 700 anti-mouse CD4 (BioLegend, San Diego, CA), PerCP/Cyanine5.5 anti-mouse CD8a (BioLegend, San Diego, CA), FITC Rat Anti-Mouse CD5 (BD Biosciences), PE Hamster Anti-Mouse CD69 (BD Biosciences), PE anti-mouse CD95 (Fas) Antibody (BioLegend, San Diego, CA), Brilliant Violet 421™ anti-mouse CD107a (LAMP-1) (BioLegend, San Diego, CA), PE anti-mouse CD137 (BioLegend, San Diego, CA).

Techniques: Marker

Journal: Frontiers in Immunology

Article Title: CD5 blockade, a novel immune checkpoint inhibitor, enhances T cell anti-tumour immunity and delays tumour growth in mice harbouring poorly immunogenic 4T1 breast tumour homografts

doi: 10.3389/fimmu.2024.1256766

Figure Lengend Snippet: Fraction of CD8 + /CD69 + T and CD4 + /CD69 + T cells after treatment with anti-CD5 Mab in vivo . (A) The fraction of CD8 + /CD69 + T and MFI of CD69 on CD8 + T cell isolated from spleens, draining lymph nodes, and TILs isolated from 4T1 tumour-bearing BALB/c mice treated with anti-CD5 MAb or isotype Mab control. (B) The fraction of CD4 + /CD69 + T and MFI of CD69 on CD4 + T cell isolated from spleens, draining lymph node, and TILs isolated from 4T1 tumour-bearing BALB/c mice treated with anti-CD5 MAb or control isotype MAb. Data are mean ± SD ( n = 3 mice), one representative experiment of three. * p < 0.05 (Student’s unpaired one-tailed t -test). MFI, mean fluorescence intensity. ns, non significant.

Article Snippet: To assess the levels of CD69, CD107a, CD137 and FasR on T cells by flow cytometry, lymphocytes obtained as described above were stained with the following antibodies: Brilliant Violet 711™ anti-mouse CD3 (BioLegend, San Diego, CA), Alexa Fluor ® 700 anti-mouse CD4 (BioLegend, San Diego, CA), PerCP/Cyanine5.5 anti-mouse CD8a (BioLegend, San Diego, CA), FITC Rat Anti-Mouse CD5 (BD Biosciences), PE Hamster Anti-Mouse CD69 (BD Biosciences), PE anti-mouse CD95 (Fas) Antibody (BioLegend, San Diego, CA), Brilliant Violet 421™ anti-mouse CD107a (LAMP-1) (BioLegend, San Diego, CA), PE anti-mouse CD137 (BioLegend, San Diego, CA).

Techniques: In Vivo, Isolation, One-tailed Test, Fluorescence

Journal: iScience

Article Title: Canonical IRE1 function needed to sustain vigorous natural killer cell proliferation during viral infection

doi: 10.1016/j.isci.2023.108570

Figure Lengend Snippet:

Article Snippet: Armenian hamster anti-mouse CD69 (clone H1.21F3), PE-Cy7 conjugated , BD Biosciences , Cat# 561930; RRID: AB_10893591.

Techniques: Blocking Assay, Virus, Recombinant, Cell Culture, Binding Assay, Isolation, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software

Journal: PLoS ONE

Article Title: A Study of T Cell Tolerance to the Tumor-Associated Antigen MDM2: Cytokines Can Restore Antigen Responsiveness, but Not High Avidity T Cell Function

doi: 10.1371/journal.pone.0000353

Figure Lengend Snippet: (A) Lymph nodes cells from 6–8 week old Ag pos and Ag neg mice were stained with antibodies against CD4, CD8 and Vβ7 and analysed by flow cytometry. Numbers in quadrants are percentage of live-gated lymphocytes. (B) Lymph node cells were stained with antibodies against CD8, Vβ7 and the activation markers CD44, CD62L, CD69 and CD25. Histograms display gated viable CD8 + Vβ7 + cells. Numbers in histograms represent the specific MFI of the activation marker staining for the gated population. Data are representative of at least three independent experiments.

Article Snippet: The following mAbs were used for flow cytometric staining: rat-anti-mouse Vβ7 FITC (Serotec, UK), rat-anti-mouse CD4 FITC, rat-anti-mouse CD8α APC, rat-anti-mouse CD8α Cy-Chrome 5 (CY-5), rat-anti-mouse Vβ7 PE, hamster-anti-mouse CD69 PE, rat-anti-mouse CD25 PE, rat-anti-mouse CD62L-FITC, rat-anti-mouse CD44 PE, rat-anti-mouse CD43 activation-associated glycoform-PE (all BD Biosciences).

Techniques: Staining, Flow Cytometry, Activation Assay, Marker

Journal: PLoS ONE

Article Title: A Quantitative Study of the Mechanisms behind Thymic Atrophy in Gαi2-Deficient Mice during Colitis Development

doi: 10.1371/journal.pone.0036726

Figure Lengend Snippet: Mature CD4 + CD8 − CD62L + CD69 − and CD8 + CD4 − CD62L + CD69 − thymocytes from wt mice and Gαi2 −/− mice were sorted by flow cytometry. DNA from the sorted thymocytes were analysed for the amount of TRECs specific DNA by real time-PCR. The frequencies of TRECs + cells in CD4 + CD62L + (white symbols) and CD8 + CD62L + (gray symbols) thymocytes were analysed in 4–9 weeks old wt mice (n = 28) and 4–8 weeks old Gαi2 −/− mice (n = 5–13), grouped based on colitis scores (rectangles = no colitis, triangles = mild colitis). The mean frequencies within each group are represented as a horizontal line. Statistical analysis was performed using the Mann-Whitney non-parametric test and values of p≤0.05 were considered significant.

Article Snippet: Mature thymocytes were purified by flow cytometry: 2×10 6 thymocytes per well in 5 wells were stained with the following antibodies: rat-anti-mouse-CD4-Phycoerythin-TexasRed (ECD) (GK1.5) (Abcam, Cambridge, UK), rat-anti-mouse-CD8-Phycoerythin-Cyanine-5 (PE-Cy5) (53-6.7) (Southern Biotech, Birmingham AL, US), hamster-anti-mouse-CD69-Phycoerythin (PE) (H1.2F3) (BD Biosciences Pharmingen, San Diego CA, US), and rat-anti-mouse-CD62L-Fluorescein isothiocyanate (FITC) (MEL-14) (BD Biosciences Pharmingen,), diluted in PBS 1% FCS up to a total volume of 30 µl and then incubated at 4°C for 20 min. For flow cytometric sorting of T lymphocytes in peripheral blood, 1–2×10 6 PBMCs in a volume of 30 µl were stained with hamster-anti-mouse-CD3-PE (145-C211) (BD Biosciences Pharmingen).

Techniques: Flow Cytometry, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: T cell kinetics reveal expansion of distinct lung T cell subsets in acute versus in resolved influenza virus infection

doi: 10.3389/fimmu.2022.949299

Figure Lengend Snippet: Formation of lung-resident CD4 + T cells in lungs after influenza virus infection. CD4 + Trm express CD69 and CD11a and to verify their localization to the lung tissue anti-CD45.2 antibodies were injected intravenously three minutes prior to euthanasia (A) . In (B) representative flow cytometry plots gated from CD4 + Tem with up to 8000 events per plot from indicated time points after infection are shown. Frequency and numbers of lung-resident CD4 + CD69 + CD11a + T cells (C) . Frequency and numbers of antigen-specific, lung-resident CD4 + CD69 + CD11a + T cells (D) . Data are composites of two separate experiments with five mice per group. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Article Snippet: The cells were stained for 30 minutes at +4°C with the following monoclonal antibodies diluted 1:50 in FACS buffer: PerCP hamster anti-mouse CD3e (145-2C11) (BD Biosciences Cat# 553067, RRID : AB_394599), V500 rat anti-mouse CD4 (RM4-5) (BD Biosciences Cat# 560782, RRID : AB_1937315)/FITC rat anti-mouse CD4 (GK1.5) (BD Biosciences Cat# 553729, RRID : AB_395013), APC rat anti-mouse CD8α (53-6.7) (BD Biosciences Cat# 553035, RRID : AB_398527), BV510 rat anti-mouse CD8α (53-6.7) (BD Biosciences Cat# 563068, RRID : AB_2687548), FITC rat anti-mouse CD8α (53-6.7) (BD Biosciences Cat# 553031, RRID : AB_394569), PE rat anti-mouse CD44 (IM7) (BD Biosciences Cat# 553134, RRID : AB_394649), FITC rat-anti mouse CD44 (IM7) (BD Biosciences Cat# 553133, RRID : AB_2076224), APC rat anti-mouse CD62L (MEL-14) (BD Biosciences Cat# 553152, RRID : AB_398533), APC-Cy7 rat anti-mouse CD62L (MEL-14) (BD Biosciences Cat# 560514, RRID : AB_10611861), V450 rat anti-mouse CD19 (1D3) (BD Biosciences Cat# 560375, RRID : AB_1645269), BV510 rat anti-mouse CD103 (M290) (BD Biosciences Cat# 563087, RRID : AB_2721775), PerCP-Cy5.5 hamster anti-rat/mouse CD49a (Ha31/8) (BD Biosciences Cat# 564862, RRID : AB_2734135), PerCP-Cy5.5 rat anti-mouse CD11a (2D7) (BD Biosciences Cat# 562809, RRID : AB_2737809), and PE-Cy7 hamster anti-mouse CD69 (H1.2F3) (BD Biosciences Cat# 552879, RRID : AB_394508), all from BD Biosciences (BD Biosciences, RRID : SCR_013311).

Techniques: Infection, Injection, Flow Cytometry