Secretome Mtb infected neutrophil and macrophage

Mtb cells was co-cultured with peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMN) in distinct conditions, then secreted proteins in the culture medium were collected and characterized using LC-MS/MS. In brief, heparinized whole blood samples of three healthy donors were provided by Srinagarind Hospital, Khon Kaen, Thailand. After isolation steps, the human leukocyte was divided into two groups: peripheral blood mononuclear cell (PBMC), and polymorphonuclear cell (PMN). PBMCs were treated with 50 nM PMA for 24 h while PMN cells were maintained in RPMI medium at 37oC. The activated PBMCs were then divided into four subgroups: PBMC alone, PBMC co-cultured with PMN after 4 h (PBMC-PMN), PBMC co-cultured Mtb (PBMC-TB), and PBMC co-cultured Mtb then added PMN after 4 h (PBMC-TB-PMN). After a total of 6 h incubation, the protein of culture supernatant was collected and measured in concentration. The protein samples were separated by SDS-PAGE and underwent in-gel digestion. Extracted protein was injected into LC-MS/MS instrument for measurement.