Cell processing tubes

Peripheral blood mononuclear cells (PBMCs) were isolated from cell processing tubes (BD Biosciences, San Jose, CA) from 3 rhesus macaques and suspended in RPMI supplemented with 10% FBS, 1% Hepes Buffer, glutamine and penicillin/streptomycin at a concentration of 10⁶ cells/mL. LtxA was added at concentrations of 1 ng/mL, 10 ng/mL, 100 ng/mL, 1 μg/mL, or 10 μg/mL, and cells were incubated for 1.5 hours at 37°C. Cells were then washed and plated in a 96-well round-bottom plate with 10⁶ cells/well. Cells were resuspended in 100 μL of Annexin V buffer (BD Biosciences) per the manufacturer’s instructions and stained with surface antibodies for 15 minutes at room temperature. The surface staining consisted of antibodies against CD3, CD4, CD8, CD56, CD11a, CD16, CD95, CD28, and CD20. The cells were then washed twice with Annexin V buffer with addition of 7AAD after the final wash and immediately acquired on the flow cytometer. Heat killed cells were used as a compensation control for 7AAD staining. For experiments using activated cells, surface staining was done prior to incubation with the same varying concentrations of LtxA, with or without addition of phorbol 12-myristate 13-acetate and ionomycin.