Briefly, cells, either THP-1 or PBMC, were seeded at 1x106/tube and left untreated or treated with autophagy inhibitors for the indicated times (i.e. 4 h or 24 h). Autophagy inhibition was obtained using different compounds: Reagent A (RA) present in the FlowCellect™ Autophagy LC3 Antibodybased Assay Kit (Merck Millipore, Burlington, MA, USA; Cat. No. FCCH100171; hereinafter referred as FlowCellect Kit) accordingly with the manufacturer’s instruction; Bafilomycin A1 (BafA1; 5μM) and Chloroquine (CQ) at the indicated concentrations (Sigma-Aldrich; Cat. No. B1793, C6628, respectively).
PBMC from active TB patients were either infected with BCG using a MOI=1 (1 bacillus/cell) (BCG Pasteur (TMC1011)), kindly provided by Prof. Giovanni Delogu (Fondazione Policlinico Universitario A. Gemelli IRCCS, Università Cattolica del Sacro Cuore, Rome, Italy),21 (link) or stimulated with 25 μg/mL of Mtb PPD (Statens Serum Institut, Copenhagen, Denmark). Treatments with BCG or PPD were carried out for 24 h either alone or in presence of CQ (20 μM) for the last 20 h.
PBMC from active TB patients were either infected with BCG using a MOI=1 (1 bacillus/cell) (BCG Pasteur (TMC1011)), kindly provided by Prof. Giovanni Delogu (Fondazione Policlinico Universitario A. Gemelli IRCCS, Università Cattolica del Sacro Cuore, Rome, Italy),21 (link) or stimulated with 25 μg/mL of Mtb PPD (Statens Serum Institut, Copenhagen, Denmark). Treatments with BCG or PPD were carried out for 24 h either alone or in presence of CQ (20 μM) for the last 20 h.