Anti cd8 pcp

Manufactured by BioLegend

Anti-CD8-PCP is a monoclonal antibody that binds to the CD8 antigen. CD8 is a glycoprotein expressed on the surface of cytotoxic T cells and a subset of natural killer cells. This antibody is conjugated with the PCP (peridinin-chlorophyll protein) fluorescent dye.

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Anti-CD8

3 protocols using anti cd8 pcp

Multi-color FACS Analysis of Murine Immune Cells

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Mouse colons and lymph nodes were processed for single cell suspension as previously published [44 (link)] and multi-color staining performed according to standard FACS staining protocols. Briefly, tissues were treated with collagenase (I, II, and IV, Sigma Aldrich, St. Louis, MO) and dispersed twice using the gentleMACs tissue dissociator (Miltenyi Biotech, Cologne, Germany). Cell suspensions were incubated in complete RPMI media for 24 hours before staining for flow cytometry. Multi-color staining was performed according to standard surface and intracellular FACS staining Biolegend protocols (Biolegend, San Diego, CA). Antibodies used in this study were anti-Ly6G-APC (clone1A8, Biolegend), anti-F4/80-PCP (PerCP/Cy5.5), (Biolegend, BM8), anti-IL-12p35-APC (eBioscience, 4010p35), anti-IL-10-FITC (Biolegend, JES5-16E3), anti-NK1.1-PCP (eBioscience, PK136), anti-CD3-FITC (Biolegend, 145-2C11), anti-CD4-PCP (Biolegend, GK1.5), anti-CD8-PCP (Biolegend, 53-6.7), anti-Tbet-FITC (Biolegend, 4B10), anti-IFNγ-PE (Biolegend, XMG1.2), anti-IL-17A-APC (Biolegend, TC11-18H10.1), and anti-IL-4-FITC (eBioscience, 11-7042-82). All samples were analyzed on a Guava easyCyte 8HT flow cytometer (EMD Millipore, Bellerica, MA, USA), and analyzed using FCS Express software (DeNovo Software, Los Angeles, CA, USA).

Morris K.T., Castillo E.F., Ray A.L., Weston L.L., Nofchissey R.A., Hanson J.A., Samedi V.G., Pinchuk I.V., Hudson L.G, & Beswick E.J. (2015). Anti-G-CSF treatment induces protective tumor immunity in mouse colon cancer by promoting NK cell, macrophage and T cell responses. Oncotarget, 6(26), 22338-22347.

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Tumor Immune Cell Profiling

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Tumors were harvested, weighed, minced, and digested for 45 minutes, with gentle shaking, at 37°C in HBSS containing 7mg/ml collagenase D and 200 μg/ml DNase-I (Roche). Single-cell suspensions were stained with antibodies including anti-CD45-APC-Cy7, anti-CD3-Vioblue, anti-CD8-PCP, anti-CD4-FITC, anti-Foxp3-PE, anti-CD11b-APC, anti-GR-1-PE-Cy7, Anti-GATA3-PE, anti-T-bet-PE-Cy7, anti-RORγt-APC (BioLegend or eBioscience); and anti-Annexin-V-Alexa647 (BD Pharmingen). Cells were fixed/permeabilized using reagents from the Foxp3 staining kit (eBioscience). Flow cytometry was performed on a Miltenyl MACS Quant10 Analyzer. Total cells were enumerated using the flow cytometer.

Steinberg S.M., Zhang P., Malik1 B.T., Boni A., Shabaneh T.B., Byrne K.T., Mullins D.W., Brinckerhoff C.E., Ernstoff M.S., Bosenberg M, & Turk M.J. (2014). BRAF-inhibition alleviates immune suppression in murine autochthonous melanoma. Cancer immunology research, 2(11), 1044-1050.

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Tumor Immune Cell Profiling

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Tumors were harvested, weighed, minced, and digested for 45 minutes with gentle shaking at 37°C, in HBSS containing 7 mg/ml Collagenase D and 2 mg/ml DNase-I (Roche). Single cell suspensions were stained with antibodies including anti-CD45-APC-Cy7, anti-CD3-Vioblue, anti-CD8-PCP, anti-CD4-PCP, anti-Foxp3-FITC, anti-CD11b-APC, CD11b-PCP, anti-Gr-1-PE-Cy7, anti-Ly6C-PE, and anti-Ly6G-PE-Cy7 (BioLegend or eBioscience); anti-CCR2-APC or control Rat IgG2B-APC antibody were obtained from R&D. Cells were fixed/permeabilized using reagents from the Foxp3 staining kit (eBioscience). Flow cytometry was performed on a Miltenyi MACSQuant 10 Analyzer. Representative gating strategies are presented in Supplementary Fig. S1. To determine absolute cell number per gram of tumor, total numbers of cells in appropriate gates were multiplied by a correction factor for the proportion of the tumor sample run through the cytometer, and normalized for total tumor weight.

Steinberg S.M., Shabaneh T.B., Zhang P., Martyanov V., Li Z., Malik B.T., Wood T.A., Boni A., Molodtsov A., Angeles C.V., Curiel T.J., Whitfield M.L, & Turk M.J. (2017). Myeloid cells that impair immunotherapy are restored in melanomas which acquire resistance to BRAF inhibitors. Cancer research, 77(7), 1599-1610.

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