Anti cd3 apc cy7 clone hit3a

Freshly isolated or cultured PBMCs were stained with Zombie Aqua™ Fixable Viability Kit (BioLegend) for 20 min at room temperature and were incubated with anti-human FcR Blocking Reagent (Miltenyi Biotec, #130–059-901) and were then incubated with cell surface antibodies for 30 min on ice. The following monoclonal antibodies were used for phenotyping of freshly isolated PBMC: anti-CD3-APC-Cy7 (clone HIT3a, Biolegend, #300317), anti-CD4-APC (clone RPA-TA, Biolegend, #300514), anti-CD8- PerCP-Cy5.5 (clone SK1, Biolegend, #344709), anti-CCR4-PE-Cy7 (clone L291H4, Biolegend, #359409), anti-CCR7-FITC (clone G043H7, Biolegend, #353215), anti-CCR10-PE (clone 314305, R&D #FAB3478P-025), anti-CD45RO-BUV395 (clone UCHL1, BD Biosciences, #564292). And the following monoclonal antibodies were used for analyzing of proliferating lymphocytes within cultured PBMC: anti-CD3-APC-Cy7, anti-CD4-PE-Cy7 (RPA-TA, Biolegend, #300512), anti-CD8-BV421 (clone RPA-T8, Biolegend, #301036). Cells were acquired with an LSR ll (BD Biosciences) and data were analyzed by using FlowJo software (FlowJo, LLC, Ashland, OR, USA).