Alexa Fluor® 647‐conjugated anti‐mouse CD4 (clone RM4‐5) and anti‐mouse CD8a (clone 53‐6.7) (BioLegend, San Diego, CA, USA, Cat.#: 100530 and 100727, respectively) were used for immunofluorescence at a final concentration of 2.5 μg·mL−1, and eFluor 660 anti‐mouse FOXP3 (clone FJK‐16 s) eBioscience™ (Carlsbad, CA, USA) (Thermo Fisher Scientific, Cat.# 50‐5773‐80) was used at 1 μg·mL−1. eFluor 660 rat IgG2a kappa Isotype Control (eBR2a) (eBioscience™, Thermo Fisher Sci., Cat. # 50‐4321‐80) and Alexa Fluor® 647 Rat IgG2a, κ Isotype Ctrl (BioLegend, Cat. #: 400526) were used as controls for staining specificity. Anti‐Granzyme B (clone D2H2F) Rabbit mAb (Cell Signaling, Danvers, MA, USA, Cat. # 17215S) and rabbit anti‐Ki67 antibody (Abcam, Cambridge, MA, USA, Cat# ab833) were used at 1 : 200 dilution. Goat anti‐rabbit AF488 (Life Technologies, Eugene, OR, USA, Cat. # A‐11008) and Texas Red‐X (Thermo Fisher Cat. # T6391) secondary antibodies were used at 1 : 500 dilution in blocking buffer.
Anti mouse cd8a clone 53 6
Manufactured by BioLegend
Sourced in United States
The Anti-mouse CD8a (clone 53-6.7) is a mouse monoclonal antibody that binds to the CD8a (Lyt-2) molecule. CD8a is a cell surface glycoprotein that is expressed on a subset of T cells, known as cytotoxic T cells, and on some natural killer (NK) cells.
Automatically generated - may contain errors
Lab products found in correlation
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Zombie aqua fixable viability kit, by BioLegend (2 mentions)
Lsrfortessa, by BD (2 mentions)
123count ebeads, by Thermo Fisher Scientific (2 mentions)
Anti mouse cd16 32 clone 93, by BioLegend (2 mentions)
Anti mouse human cd44 clone im7, by BioLegend (2 mentions)
Anti mouse cd62l clone mel 14, by Thermo Fisher Scientific (2 mentions)
Anti mouse human granzyme b clone gb11, by BioLegend (2 mentions)
Anti mouse ctla4 clone uc1 4b9, by BioLegend (2 mentions)
Anti mouse cd25 clone pc61, by Thermo Fisher Scientific (2 mentions)
Anti mouse ifn γ clone xmg1, by BioLegend (2 mentions)
Anti mouse cd45 clone 30 f11, by BioLegend (2 mentions)
Anti mouse ly6c clone hk1, by BioLegend (2 mentions)
Anti mouse cd4 clone gk1, by BioLegend (2 mentions)
Goat anti rabbit af488, by Thermo Fisher Scientific (1 mentions)
Rabbit anti ki67 antibody, by Abcam (1 mentions)
Texas red x, by Thermo Fisher Scientific (1 mentions)
A 21244, by Thermo Fisher Scientific (1 mentions)
Gtx127309, by GeneTex (1 mentions)
Anti mouse cd4 clone rm4 5, by BioLegend (1 mentions)
7 protocols using anti mouse cd8a clone 53 6
1
Immunofluorescence Staining of Immune Cells
2
Murine Skin Immunofluorescence Staining
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For murine skin IF samples, immediately after sacrifice, mouse skin tissues were fixed with periodate-lysine-paraformaldehyde fixative overnight. Samples were then dehydrated with 30% sucrose, embedded in OCT compound (Tissue-Tek), and stored at −80°C. Sections were cut at 7 μm and stained with the following Abs: anti–mouse CD4 (clone RM4-5; BioLegend, catalog 100547; 1:100), anti–mouse CD8a (clone 53-6.7; BioLegend, catalog 100747; 1:100), anti-pimonidazole mouse FITC- or DyLight 549-Mab (clone 4.3.11.3; Hypoxyprobe, catalog HP6-200 or HP7-200; 1:100), rabbit polyclonal anti–HIF-1α (GeneTex, catalog GTX127309; 5 μg/mL) or rabbit polyclonal isotype control (GeneTex, catalog GTX35035; 5 μg/mL), and Alexa Fluor 647–conjugated goat anti-rabbit secondary Ab (Thermo Fisher Scientific, catalog A-21244; 1:1,000). Confocal microscopy was conducted with a Leica SP8 (Figure 1 and Supplemental Figure 2 ) or SP5 (Supplemental Figure 3 ) laser-scanning confocal microscope at the Cell Imaging Core, Yale Stem Cell Center.
3
Multiparametric Flow Cytometry of CD8+ T Cells
4
Phenotyping Liver Non-Parenchymal Cells
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Non-parenchymal liver cells were incubated with monoclonal antibody 2·4G2 for FcR blocking (BioLegend, San Diego, CA, USA) and then exposed at 4°C to a mixture of the following antibodies (dilutions are indicated):anti-mouse CD45 (clone 30-F11, 1:100), anti-mouse/human CD11b (clone M1/70, 1:300), anti-mouse Ly6C (clone HK1.4, 1:300), anti-mouse MHCII (clone M5/114.15.2, 1:200), anti-mouse CD11c (clone N418, 1:100), anti-mouse CD3ϵ (clone 145-2c11, 1:100), anti-mouse CD8a (clone 53-6.7, 1:100), anti-mouse CD4 (clone GK1.5, 1:100), anti-mouse TCRβ (clone 457-597, 1:100) all were purchased from BioLegend, San Diego, CA. Anti-mouse F4/80 (clone REA 126, 1:100) and anti-mouse Tim4 (clone REA999, 1:100) were purchased from Miltenyi Biotech.
For IL-17A staining, cells were first stained for surface markers, then the cells were fixed and permeabilized prior to intra-cellular staining with IL-17 (Clone TC11-18H10.1, 1:50, BioLegend). Cells were analyzed with BD FACS Canto™ II (BD Bioscience) or sorted with a FACSAria flow cytometer (BD Bioscience). Flow cytometry analysis was performed using FlowJo software (TreeStar, Ashland, OR).
For IL-17A staining, cells were first stained for surface markers, then the cells were fixed and permeabilized prior to intra-cellular staining with IL-17 (Clone TC11-18H10.1, 1:50, BioLegend). Cells were analyzed with BD FACS Canto™ II (BD Bioscience) or sorted with a FACSAria flow cytometer (BD Bioscience). Flow cytometry analysis was performed using FlowJo software (TreeStar, Ashland, OR).
5
Multiparametric Flow Cytometry of CD8+ T Cells
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FC receptors were blocked with anti-mouse CD16/32 (clone 93, Biolegend) before staining. Dead cells were identified using the Zombie Aqua Fixable Viability Kit (Biolegend). Cells were stained with the following antibody clones: anti-mouse CD8a (clone 53-6.7, Biolegend), anti-mouse/human CD44 (clone IM7, Biolegend), anti-mouse CD62L (clone MEL-14, eBioscience), anti-mouse/human Granzyme B (clone GB11, Biolegend), anti-mouse CTLA4 (clone UC1-4B9, Biolegend), anti-mouse CD25 (clone PC61.5, eBioscience), and anti-mouse IFN-γ (clone XMG1.2, Biolegend). Intracellular staining of Granzyme B, CTLA-4, and IFN-γ was performed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). To count cells, 123count eBeads (eBioscience) were added to flow cytometry tubes immediately before flow cytometer acquisition. Data was acquired on a BD LSRFortessa and analyzed in FlowJo. Cells were gated for size, single cells, living cells, and CD8+ cells before examination of proliferation curves (Supplementary Fig. 8c ). Cells were further gated on proliferation dye+ cells to exclude any CD8+ T cells in the APC fraction before quantification of division numbers and examination of surface and intracellular proteins. Statistical analyses of results from separate mice were performed using GraphPad Prism software.
6
Flow Cytometric Analysis of Lymphocyte Subsets
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Lymph nodes were homogenized in RPMI-1640 culture medium with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin, 2 mM L-glutamine, 1x MEM non-essential amino acids (Gibco, USA), 1 mM sodium pyruvate (Corning, USA), 10 mM HEPES (Fisher Scientific, USA), and 40 µM 2-mercaptoethanol (Sigma Aldrich) in gentleMACS C tubes (Miltenyi, USA). 0.5x106 cells were incubated for 4 h with 20 ng/ml phorbol 12-myristate 13-acetate (PMA), 1 µg/ml ionomycin (Sigma Aldrich), and 3 µg/ml brefeldin A (Invitrogen, USA).
Subsequently, cells were washed with stain buffer containing bovine serum albumin (BD Pharmingen, USA) and stained with anti-mouse CD4 (clone GK1.5) and anti-mouse CD8a (clone 53-6.7, BioLegend, USA). Cells were then fixed, washed, and stained with anti-mouse IFN-γ antibody (clone XMG1.2, BioLegend), anti-mouse IL-4 antibody (clone BVD4-1D11, Miltenyi), or anti-mouse IL-17A antibody (clone TC11-18H10.1, BioLegend). Cells were analyzed on a Novocyte 3005 flow cytometer (Acea Biosciences, USA). The following gating strategy was used in FlowJo software v.10.6.0 (FlowJo, USA): size (FSC-H vs. SSC-H) → single cells (FSC-H vs. FSC-A) → CD8a+ vs CD4+ → IFN-γ+ (Th1), IL-4+ (Th2), or IL-17A+ (Th17) of the CD4+CD8- population.
Subsequently, cells were washed with stain buffer containing bovine serum albumin (BD Pharmingen, USA) and stained with anti-mouse CD4 (clone GK1.5) and anti-mouse CD8a (clone 53-6.7, BioLegend, USA). Cells were then fixed, washed, and stained with anti-mouse IFN-γ antibody (clone XMG1.2, BioLegend), anti-mouse IL-4 antibody (clone BVD4-1D11, Miltenyi), or anti-mouse IL-17A antibody (clone TC11-18H10.1, BioLegend). Cells were analyzed on a Novocyte 3005 flow cytometer (Acea Biosciences, USA). The following gating strategy was used in FlowJo software v.10.6.0 (FlowJo, USA): size (FSC-H vs. SSC-H) → single cells (FSC-H vs. FSC-A) → CD8a+ vs CD4+ → IFN-γ+ (Th1), IL-4+ (Th2), or IL-17A+ (Th17) of the CD4+CD8- population.
7
Murine Immune Cell Profiling
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Murine samples of spleen tissues, lymph nodes and tumors were collected and prepared as previously described (23) . Cells were stained with the following antibodies: antimouse CD3 (clone 17A2, BioLegend, Cat#100221, RRID:AB_2057374), antimouse CD4 (clone GK1.5, Miltenyi Biotec, Cat#130-121-131, RRID:AB_2752219), antimouse CD8a (clone 53-6.7, BioLegend, Cat#100712, RRID:AB_312751), antimouse CD45 (clone 30-F11, BioLegend, Cat#103108, RRID:AB_312973, BD Biosciences, Cat#557659, RRID:AB_396774), antimouse F4/80 (clone BM8, BioLegend, Cat#123115, RRID:AB_893493), antimouse CD11b (clone M1/70, Miltenyi Biotec, Cat# 130-097-585, RRID:AB_2660136), antimouse Gr-1 (clone RB6-8C5, TONBO, San Diego, CA), antimouse Ly6C (clone HK1.4, BioLegend, Cat#128006, RRID:AB_1186135). Data was acquired using MACS Quant flow cytometer (Miltenyl Biotec, Bergisch Galdbach, Germany) and analyzed using FlowJo software (FlowJo, RRID:SCR_008520).
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