Apc cy7 labelled anti cd8

To examine the expression of multiple cytokines (perforin, granzyme B, IFN-γ) and checkpoint receptor members ((programmed cell death protein 1 (PD-1) and T-cell Ig and mucin domain 3 (Tim-3)) in the antigen-specific CD8⁺ memory T-cell subsets, PBMCs were isolated by density gradient centrifugation and resuspended in phosphate buffer saline (PBS) containing 2% fetal calf serum. Thereafter, CD8⁺ T_CM, T_EM, and T_EMRA were isolated with allophycocyanin (APC)-labelled Pro5MHC Pentamer [HLA-A*03:01 MOG_46-55 pentamers-RVVHLYRNGK] (Proimmune, Oxford, UK), phycoerythrin (PE)-cy7-labelled anti-CCR7 (eBioscience, San Diego, CA), APC-cy7-labelled anti-CD8 (BD Pharmingen, San Diego, CA, USA), and peridinin chlorophyll protein (PerCP)-Cy5.5-labelled-anti-CD45RA (Tonbo Biosciences, San Diego, CA) monoclonal antibody (MoAb) via a flow sorter (Becton Dickinson, San Jose, CA, USA).

Fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, allophycocyanin (APC)-, BD Horizon™ V450 (V450)-, and PE-Cy5-labelled anti-CD3 (UCHT1); APC-labelled anti-CD27 (L128); FITC-labelled anti-CD19 (HIB19); APC-labelled anti-CD45RO (UCHL1); APC-labelled anti-CD19 (HIB19); FITC-labelled anti-CD56 (MCAM16·2); Alexa Fluor® 700-labelled anti-CD4 (RPA-T4); APC-Cy™7-labelled anti-CD8 (SK1); APC-Cy™7-labelled anti-CD69 (FN50); FITC-labelled anti-CD95 (DX2); PE-Cy7-labelled anti-CD3 (SK7); PE-labelled anti-CD45RA (HI100); FITC-labelled anti-CD28 (CD28.2); FITC-labelled anti-CD94 (HP-3D9); FITC-labelled anti-T-cell receptor (TCR) αβ (WT31); PE-labelled anti-TCR αβ (T10B9.1A-31); FITC-labelled anti-CD69 (FN50); PE-Cy7-labelled anti-CCR7 (3D12); BD Horizon™ V500 (V500)-labelled anti-CD8 (RPA-T8); and 7-amino-actinomycin D (7-AAD) were purchased from BD Biosciences (Franklin Lakes, NJ). Pacific Blue™-labelled anti-CD107a (LAMP-1) was purchased from Biolegend (San Diego, CA). PE-labelled anti-TCR γδ (B1.1) was purchased from eBioscience (San Diego, CA). FITC-labelled anti-TCR pan γδ (IMMU510) was purchased from Beckman Coulter (Fullerton, CA). Pacific Orange-labelled anti-CD8 (3B5) was purchased from Invitrogen (Camarillo, CA).