Anti cd8 apc clone sk1

PBMCs were suspended at 1 x 10⁶/mL in PBS and incubated at 37^°C for 20 min with 0.5 uM carboxyfluorescein succinimidyl ester (CFSE; Life Technologies). After the addition of serum and washes with PBS, cells were resuspended at 1 x 10⁶/mL and plated into 96-well U-bottom plates (Corning) at 200 uL volumes. Peptide pools were added at a final concentration of 0.25 ug/mL. On day 6, cells were harvested, washed with PBS + 2% Fetal Bovine Serum, and stained with anti-CD3-PE-Cy7 (clone SK7; BioLegend), anti-CD8 APC (clone SK1; BioLegend), anti-CD4 BV711 (clone RPA-T4; BioLegend) and LIVE/DEAD violet viability dye (Life Technologies). Cells were washed and fixed in 2% paraformaldehyde, prior to flow cytometric analysis on a BD LSR II (BD Biosciences). A positive response was defined as one with a percentage of CD3⁺ CD8⁺ or CD3⁺ CD4⁺ CFSE low cells at least 1.5x greater than the highest of two negative-control wells and greater than 0.2% CD8⁺ or CD4⁺ CFSE low cells in magnitude following background subtraction. For graphical analyses, responses are plotted at a value of 0.1% CD8⁺ or CD4⁺ CFSE low cells.

Flat-bottom 96 well plates were prepared with or without anti-CD3 coating at 1 μg/mL (clone OKT3, catalog 14-0037-82, eBioscience). hCTLs were stained with 10 μM cell proliferation dye eFluor 450 (65-0842-85, Thermo Fisher) for 10 minutes in the dark at 37°C, followed by quenching with FBS-containing media for 5 minutes. Subsequently, hCTLs were washed 3× with FBS-containing media, and 20,000 hCTLs were seeded per well. After 5 days, cells were washed 2× in ice-cold D-PBS (Thermo Fisher) prior to staining with an anti–CD8-APC (clone SK1, catalog 344722, BioLegend) or anti-CD4-PE (clone A161A1, catalog 357404, BioLegend) and Live/Dead fixable yellow (catalog L34959, Thermo Fisher). hCTLs were washed in PBS, 1% FCS prior to analysis on a LSR Fortessa flow cytometer. Samples were gated on live CD8⁺ cells; results were analyzed using FlowJo 10 software.