Anti cd69 pe cy5

We thawed PBMCs from cryovials and washed them as described above and seeded 2.5 × 10⁶ cells/ml in RPMI 1640 with 10% FBS or 2% Phx in five separate wells corresponding to time of incubation. We performed extracellular staining with fixable viability dye eFluor 780 (eBioscience), anti-CD3-PerCP-cyanine 5.5 (Cy5.5) (BioLegend), anti-CD8-BV605 (BioLegend), anti-CD4-BV510 (BioLegend), anti-Vα7.2-PE-Cy7 (BioLegend), anti-LAG-3-BV786 (BioLegend), anti-CD69-allophycocyanin (BioLegend), anti-CD25-PE-Cy5 (BD Biosciences), anti-PD-1 Alexa Fluor 700 (BioLegend), anti-CD161-BV605 (BioLegend), anti-CD69-PE-Cy5 (BioLegend), and anti-human PE-MR1–5-OP-RU tetramer (National Institutes of Health [NIH] Tetramer Core Facility). We collected data using a five-laser LSRFortessa flow cytometer (BD Biosciences), and the flow data were analyzed using FlowJo software version 10 (BD Biosciences, Ashland, OR).

Using a 7-color flow cytometry assay, 1×10⁶ fresh, frozen/not rested and frozen/rested overnight PBMC were analyzed for phenotype and the activation marker, CD69. To determine viability, PBMC were washed twice with PBS, resuspended and incubated in 1μL of Aqua amine-reactive viability dye (Molecular Probes, Inc. Eugene, OR) for 20 min at RT in the dark, washed and incubated with 10μL of Fc block (Miltenyi Biotec Inc., Auburn, CA) for another 20 min at RT in the dark. The cells were washed with FACS buffer (0.2% BSA+0.1% NaN₃ in PBS) and incubated for 20 min in the dark at 4°C with anti-CD3-eFluor 450, anti-CD19-APCeFluor 780 (both from eBioscience), anti-CD56-PE/Cy7, anti-CD14-APC, anti-CD16-FITC and anti-CD69-PE/Cy5 (Biolegend, San Diego, CA). After incubation, cells were washed in FACS buffer and fixed in 1% paraformaldehyde. Duplicate sets of PBMC: fresh, frozen/thawed and frozen/thawed/rested overnight, were incubated overnight with or without IL-2 and were stained with identical cocktails or in the absence of anti-CD69 (supplemental Figure 1) for fluorescence minus one (FMO) control (Supplemental Figure 1). Samples were analyzed using an LSRII flow cytometer and FlowJo software (TreeStar Inc., Ashland, OR). Dead cells were excluded from analysis by gating on Aqua amine-reactive viability dye negative cells.