Whole Blood Assay for Tuberculosis

Blood was collected in sodium heparin tubes and processed within 3 hours of collection. The whole blood assay was adapted from the protocol described by Hanekom et al.^{46 (link)}. Briefly, 0.5 ml of whole blood was stimulated with a pool of 300 Mtb-derived peptides (Mtb300, 2 µg/ml)^{47 (link)} at 37°C for 5 hours in the presence of the co-stimulatory antibodies, anti-CD28 and anti-CD49d (1 μg/ml each; BD Biosciences) and Brefeldin-A (10 µg/ml; Sigma-Aldrich). Unstimulated cells were incubated with co-stimulatory antibodies and Brefeldin-A only. Red blood cells were then lysed in a 150 mM NH₄Cl, 10 mM KHCO₃, 1 mM Na₄EDTA solution. Cells were stained with a Live/Dead Near-InfraRed dye (Invitrogen) and then fixed using a Transcription Factor Fixation buffer (eBioscience), cryopreserved in freezing media (50% fetal bovine serum, 40% RPMI and 10% dimethyl sulfoxide) and stored in liquid nitrogen until use.