Stimulation of PBMCs with mycobacterial antigens
Five ml of blood was used for isolation of peripheral blood mononuclear cells (PBMCs). Briefly, PBMCs were separated using Ficoll-Histopaque as described (26) and plated at 106 cells/ well in a 24 well plate. Cells were incubated with purified protein derivative antigen (PPD) at 10 µg/ml (BeiResources, Colorado State University, USA) or left unstimulated for 18h at 37oC. Cellular supernatants were collected after centrifugation. Cells were washed and the pellets were snap-frozen at -80°C until tested. Cell monolayers were lysed and harvested using TriZol reagent (Invitrogen, USA) and stored at -70°C until processed further.