A700 anti human cd4 antibody

For T cell isolation, blood samples were diluted with 1 volume of PBS 2% FBS, then the diluted blood was added dropwise to 1 volume of Lymphoprep (Stemcell Technologies Inc, Cat# 07811) at room temperature. Samples were centrifuged at 800g for 20 minutes at 22 °C, then the PBMCs layer was harvested, washed two times in PBS 2% FBS, and resuspended in PBS 2% FBS. CD8+ T cells were then purified by two subsequent rounds of isolation: first, T cells were enriched using the Pan T Cell Isolation kit (Miltenyi Biotec, Cat# 130-096-535); then, negative CD8 T cell isolation was performed with the CD8+ T Cell Isolation Kit, human (Miltenyi Biotec, Cat# 130-096-495) or negative CD4 T cell isolation was performed with the CD4+ T Cell Isolation Kit, human (Miltenyi Biotec, Cat# 130-096-533). Both enrichment steps were performed using an AutoMACS machine. For both mouse and human cells, CD8 T cell purity was assessed by FACS staining using mouse or human anti-CD3 and anti-CD8 antibodies at 1:100 dilution (PE/Cyanine7 anti-human CD3, BioLegend, # Cat317333; APC/Cyanine7 anti-mouse CD8a, BioLegend, Cat# 100714; FITC anti-human CD3, ThermoFisher Scientific, Cat#11-0038-42; PE/Cy7 anti-human CD8, Biolegend, Cat# 344712). Human CD4+ T cell purity was assessed using the A700 anti-human CD4 antibody, Biolegend, Cat# 317426 at 1:100 dilution.