Quantifying Mycobacterial Antigen Release from Infected Dendritic Cells

Bone marrow-derived dendritic cells (BMDC), generated as previously described (20 (link)), were seeded in 96-well tissue culture-treated plates (Corning) at 2 × 10⁶ cells/well, rested for 2 h, infected overnight at different multiplicities of infection (1, 2, 4, and 8) with M. tuberculosis H37Rv, treated with amikacin (200 µg/ml for 40 min) in BMDC medium (RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum [FBS], 2 mM l-glutamine, 1 mM sodium pyruvate, 1× β-mercaptoethanol, 10 mM HEPES, and 12 ng/ml recombinant mouse granulocyte-macrophage colony-stimulating factor [GM-CSF]), washed three times in PBS, and further cultured in fresh BMDC medium. Conditioned medium (CM) was harvested 16, 24, 34, and 48 h later and sterile filtered, and Ag85B in CM was quantified by a sandwich ELISA. At each harvest time point, infected BMDC were assayed for cell death by using CellTiter-Glo (Promega) according to the manufacturer’s instructions, and the signal was read as luminescence with a Synergy H1 microplate reader (BioTek). For each harvest time point, the signal from uninfected cells was considered 100% viability for determination of loss of viability of infected cells.