Cd8a percp cy5

Manufactured by BioLegend

CD8a-PerCp-Cy5.5 is a fluorophore-conjugated antibody that binds to the CD8a protein. CD8a is a transmembrane glycoprotein expressed on the surface of T cells, thymocytes, and some natural killer cells. The PerCp-Cy5.5 fluorophore is conjugated to the antibody, allowing for the detection and analysis of CD8a-positive cells using flow cytometry.

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Close spelling variants of this product

Anti-CD8a PerCP/Cy5.5 (HIT8a), CD8a-PerCP-Cy5.5 (53–6.7; Anti-CD8a-PerCP/Cy5.5 PerCP/Cy5.5-CD8a Percp/Cy5.5 anti-mouse CD8a PerCP-Cy5.5–anti-CD8a CD8a-PerCP PerCP-Cy5.5

5 protocols using cd8a percp cy5

Dissecting Murine Lymphoid Cell Subsets

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Lymphoid cells from bone marrow, thymus and spleen were isolated from 8-week old mice. To reveal T cell progenitor subsets in the thymus, single cell suspensions were stained with conjugated antibodies, specific for CD3-FITC (Biolegend), CD4-APC, CD8a-PerCp-Cy5.5, CD25-PE, CD44-APCCy7 (Biolegend), TCRβ-Pacific Blue (Biolegend). To analyse lymphoid subsets in the spleen, single cell suspensions were stained with CD3-FITC, CD4-APC, CD8a-PerCp-Cy5.5, CD19-APCH7, CD45R(B220)-PacificBlue, IgD-PE (eBioscience), IgM-PECy7 (eBioscience). To define the B cell progenitor subsets, suspensions from the bone marrow were stained with IgD-FITC, CD25-PE, IgM-PECy7 (eBioscience), CD45R (B220)-PacificBlue, CD117 (cKit)-APC (eBioscience), CD19-APCH7. Dead cells were excluded from the analysis by propidium iodide staining. Antibodies were purchased from BD Pharmingen unless mentioned otherwise. Samples were measured on a FACS Fortessa^® and analysed using FlowJo^® software (Version: 10.0.8r1).

Pilzecker B., Buoninfante O.A., Pritchard C., Blomberg O.S., Huijbers I.J., van den Berk P.C, & Jacobs H. (2016). PrimPol prevents APOBEC/AID family mediated DNA mutagenesis. Nucleic Acids Research, 44(10), 4734-4744.

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Tumor Immune Microenvironment Profiling

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To characterize the subpopulations of the tumor immune microenvironment, tumors were obtained at the end of study. Tumors were isolated and minced into small pieces and digested in RPMI medium containing collagenase D (1 mg/ml; Roche) and DNase1 (150 UI/ml; Sigma) for 30 minutes at 37°C and then washed and filtered twice using 100 µm cell strainers (Falcon^®). Fixable Viability Stain 620 (BD Biosciences) was used to discriminate live and dead cells. The cells were then blocked with Fc-block (BD Biosciences) and stained with anti-mouse antibodies against: CD3 (FITC; Biolegend), CD4 (PERCP/Cy5.5 or AF647; Biolegend), CD8a (PERCP/Cy5.5; Biolegend), IFN-γ (AF488; Biolegend), CD25 (APC; Biolegend), Foxp3 (PE; Biosciences), and IL-4 (APC; Biosciences), as per the manufacturer's instructions. Stained samples were analyzed on Cyan ADP 9 colors cytometer (Beckman Coulter) and analyses were performed with FlowJo software version 10.

Zhang S.L., Mao Y.Q., Zhang Z.Y., Li Z.M., Kong C.Y., Chen H.L., Cai P.R., Han B., Ye T, & Wang L.S. (2021). Pectin supplement significantly enhanced the anti-PD-1 efficacy in tumor-bearing mice humanized with gut microbiota from patients with colorectal cancer. Theranostics, 11(9), 4155-4170.

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Multiparametric Flow Cytometry of Murine Immune Responses

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Single suspension cells from spleens and lymph nodes collected 14 days after the second immunization were prepared and stimulated with 10 μg/ml peptides at 37°C for 5 h. Cells were stained with viability dye eflour780 in PBS for 15 minutes on ice followed by washes twice with PBS supplemented with 2% FBS. To detect cell surface antigens, cells were stained with fluorochrome-tagged antibodies, as shown in the following table for 15 minutes on ice. To detect intracellular cytokines or intranuclear transcription factors, cells were fixed and permeabilized using an intercellular cytokine staining kit (Biolegend) or a commercial transcription factor staining kit (eBioscience). All stained samples were run on LSRFortessa (Biolegend) and analyzed by FlowJo (TreeStar).

Antibody	Company	Clone	Lot number
anti-Mouse CD4-APC	eBioscience	GK1.5	4329627
anti-Mouse CD8a-PerCP/Cy5.5	Biolegend	53–6.7	B219152
anti-Mouse TNFα-PE	eBioscience	MP6-XT22	438513
anti-Mouse Granzyme B-PE Cyanine7	eBioscience	NGZB	4281151
anti-Mouse IFNγ-APC	eBioscience	XMG1.2	4289683
anti-Mouse IFNγ-BV421	Biolegend	XMG1.2	B232596
anti-Mouse CD3e-FITC	eBioscience	145–2C11	E00061-1632
anti-Mouse IL-5-PE	eBioscience	TRFK5	12–7052-82
anti-Mouse IL-13-eFlour710	eBioscience	eBio13A	46–7133-82

Jiang S., Wu S., Zhao G., He Y., Bao L., Liu J., Qin C., Hou J., Ding Y., Cheng A., Jiang B., Wu J., Yan J., Humeau L., Patella A., Weiner D.B., Broderick K, & Wang B. (2022). Comparison of Wild Type DNA Sequence of Spike Protein from SARS-CoV-2 with Optimized Sequence on The Induction of Protective Responses Against SARS-Cov-2 Challenge in Mouse Model. Human Vaccines & Immunotherapeutics, 18(1), 2016201.

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Comprehensive Immune Cell Profiling

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Antibodies specific for: CD-8a-488 (clone 53-6.7 Biolegend), IL-2-488 (clone JES6-5H4 eBioscience), CD169-488 (clones SER4 and MOMA-1 in house), CD11a-FITC (clone M17/4 eBioscience), CD11c (clone N418 eBioscience), CD44-FITC (clone HI44a ImmunoTools), CD11c-PE (clone N418 eBioscience), CD38-PE (clone 90 eBioscience), CD4-PE (clone GK1.5 eBioscience), CD8a-PE (clone 53-6.7 eBioscience), GL7-biotin (eBioscience), B220-ef450 (clone 6B2 eBioscience), KLRG1-ef450 (clone 2F1 eBioscience), CD127/IL7Rα-APC (clone A7R34 Biolegend), CD8a-APC (clone 53-6.7 eBioscience), IFNγ-APC (clone xM61.2 eBioscience), CD62L-PECy7 (clone MEL-14 Biolegend), CD8a-PECy7 (clone 53-6.7 eBioscience), CD4-PERCPCy5.5 (clone RM4-5 eBioscience), CD8a-PERCPCy5.5 (clone 53-6.7 Biolegend). OVA-488 (Invitrogen). H-2Kb-SIINFEKL Tetramers (LUMC, Leiden). LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit (Invitrogen) was used according to manufacturers' protocol.

van Dinther D., Veninga H., Revet M., Hoogterp L., Olesek K., Grabowska J., Borg E.G., Kalay H., van Kooyk Y, & den Haan J.M. (2018). Comparison of Protein and Peptide Targeting for the Development of a CD169-Based Vaccination Strategy Against Melanoma. Frontiers in Immunology, 9, 1997.

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Isolation and Analysis of Tumor-Associated Immune Cells

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The carcinogen 4-nitroquinoline-1-oxide (4NQO) was purchased from Sigma–Aldrich and diluted in sterile water to a final concentration of 50 μg/mL or 100 μg/mL. The in vivo anti-mouse Ly6G/Ly6C (Gr1) neutralizing antibody was obtained from BioXcell. Palmitic acid (PA), oleic acid (OA), and oil red O were provided by Sigma–Aldrich. DNase I was acquired from Biofroxx. FcR-Blocking Reagent (anti-mouse CD16/32), Zombie Green^TM Fixable Viability Kit and the following fluorescently labeled antibodies were from BioLegend: anti-mouse mAbs CD45-BV605, Ly6G/Ly6C (Gr1)-BV510, Gr1-AF594, Ly6C-BV421, Ly6G-PE, CD3-APC/Cy7, CD4-PE/Cy7, CD8a- PerCP/Cy5.5, CCR1 (CD191)-APC, CD11b-APC, and CD11b-AF700. RBC lysis buffer, IC fixation buffer, permeabilization buffer (10×), and anti-mouse mAbs Arg1-APC and iNOS-PE were obtained from eBioscience. The CellTrace™ Violet Cell Proliferation Kit, BODIPY^TM 493/503, and immunostaining antibody against CCR1 were from Life Technologies. Rabbit anti-mouse Ki67 antibody was purchased from Arigo Biolaboratories. Primary anti-mouse/human CD11b, GAPDH, anti-human CD33, and anti-mouse MIP1γ (CCL9) antibodies were purchased from Abcam. Immunostaining antibody against Ly6G was acquired from Servicebio. CCL9 protein was from NovoProtein. Secondary antibodies coupled to fluorescent Alexa probes were purchased from Beijing Emarbio Science Technology.

Peng J., Hu Q., Chen X., Wang C., Zhang J., Ren X., Wang Y., Tao X., Li H., Song M., Cheng B., Wu T, & Xia J. (2021). Diet-induced obesity accelerates oral carcinogenesis by recruitment and functional enhancement of myeloid-derived suppressor cells. Cell Death & Disease, 12(10), 946.

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