Cd8 pe cy7 clone sk1

The following anti-human antibodies were used for the flow cytometry assays: CD3 A700 (clone SK7; BioLegend, San Diego, CA), CD8 PE-Cy7 (clone SK1; BioLegend) PD-1 FITC (clone EH12.2H7; BioLegend), LAG-3 PerCPCy5.5 (clone 11C3C65; BioLegend), For the surface stain, mAbs against cell markers were added to a total of 1 × 10⁶ cells in 1X PBS containing 1% FBS and 0.1% sodium azide (FACS buffer) for 45 min at 4°C. After washing twice with FACS buffer, cells were fixed in PBS containing 2% paraformaldehyde (Sigma-Aldrich, St. Louis, MO). For each sample, 100,000 total events were acquired on the BD LSRII. Ab capture beads (BD Biosciences) were used as individual compensation tubes for each fluorophore in the experiment. To define positive and negative populations, we used fluorescence minus controls for each fluorophore. Furthermore, we optimized gating by examining known negative cell populations for background expression levels similar to that used in our previous work (7 (link)). Briefly, we gated single cells, dump cells, viable cells (Aqua Blue), lymphocytes, CD3⁺ cells, and CD8⁺ cells before finally gating human epitope-specific CD8⁺ T cells using HSV-specific tetramers (Figure S1). Data analysis was performed using FlowJo software (BD Biosciences, San Jose, CA). Statistical analyses were done using GraphPad Prism version 5 (La Jolla, CA).