V500 mouse anti human cd8

1 × 10⁵ tumor cells were incubated with CFSE-labeled PBMCs (E:T = 10:1) in medium supplemented with TfR-BiTE at the concentrations indicated for 48 h. Thereafter, the cell mixture was cultured in the medium without TfR-BiTE for an additional 72 h. Then cells were stained with a mixture of V450 mouse anti-human CD3, APC-Cy7 mouse anti-human CD4, and V500 mouse anti-human CD8 (BD Biosciences). After washing, the cells were stained with 7-AAD for 15 min. Proliferation on 7-ADD⁻/CD3⁺/CD4⁺ and 7-ADD⁻/CD3⁺/CD8⁺ T-cell subsets was determined by flow cytometry.

Human PBMCs were isolated from whole blood using lymphoprep density gradient solution (Stemcell Technologies). CD8⁺ T cells were purified from PBMCs using CD8 MicroBeads (Miltenyi Biotec). Antigen-presenting cells (APCs) were isolated from PBMCs by depleting CD4⁺ and CD8⁺ cells. APCs were pulsed with flu peptide (CEF1 influenza matrix protein M1 (58-66) (RP19978, GenScript)) at 15 nM for 8 hrs. Isolated CD8 T (negative isolation) cells were cocultured (5 : 1 ratio) with antigen-pulsed APCs for 72 h. CD8⁺ cells were subjected to tracer uptake and flow cytometry [9 (link)]. The follwoing antibodies were used for flowcytometry: CD3 Monoclonal Antibody (UCHT1), APC-eFluor 780, eBioscience (47-0038-420), V500 Mouse Anti-Human CD8, BD Biosciences (560774) SAMHD1 Recombinant Rabbit Monoclonal Antibody (JU56-04), Invitrogen (MA5-32810), Alexa Fluor® 647 Donkey anti-rabbit I (406414), FITC anti-human CD69 Antibody (310904), PE anti-human CD279 (PD-1) Antibody (367404).