Iscove s modified eagle medium imdm

Spleens were isolated aseptically and single-cell suspensions made in complete media comprising Iscove’s Modified Eagle Medium (IMDM) (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/mL penicillin G and 100 μg/mL streptomycin. Single-cell suspensions were achieved by passing the organs through a 40-μm nylon cell strainer (Becton Dickson, NJ) using a 2-mL syringe plunger. Cells were then spun at 1200 rpm for 5 min and media discarded, and the red blood cells lysed by resuspending in 1 mL RBC lysis buffer (8.34 g ammonium chloride, 0.037 g EDTA and 1 g sodium hydrogen carbonate/L, pH 7.2) for 1 min. Cells were pelleted again and resuspended in complete media. Viability was determined by trypan blue exclusion. Cells were then reconstituted to a working concentration of 10⁷ cells/mL and used for culture and flow cytometry. Cells were plated in a 96-well plate and stained for expression of extracellular markers.

The murine macrophage cell line J774.1 (American Type Culture Collection, TIB-67) was maintained at 37°C and 5% CO₂ in Dulbecco’s modified Eagle’s medium (Thermo Scientific, Rockford, IL, USA) supplemented with 10% (v/v) FBS, 2 mM glutamine, and essential amino acids. BMDCs were generated from mouse BM as previously described (40 (link), 61 (link)) and maintained at 37°C and 5% CO₂ in Iscove’s modified Eagle medium (IMDM; Gibco Invitrogen, UK) supplemented with 10% FBS (Gibco Invitrogen), recombinant mouse GM-CSF (1.5 ng/ml; PeproTech, Rocky Hill, NJ, USA), mouse IL-4 (1.5 ng/ml; PeproTech, USA), penicillin (100 units/ml; Gibco Invitrogen), streptomycin (100 µg/ml; Gibco Invitrogen), gentamicin (50 µg/ml; Gibco Invitrogen), l-glutamine (2 mM; Gibco Invitrogen), and β-mercaptoethanol (50 nM; Gibco Invitrogen). The J774A.1 cells and BMDCs were infected with the rBCG strains, rBCG-pMyong2-p24, -pAL-p24, and -pMV306-p24 and wild type BCG strains (10 M.O.I.) (in triplicate) for 4 h, followed by three washes with PBS and culturing for 24 h with fresh media. After 24 h, the infected cells were lysed with 0.5% Triton X-100. The cell lysates were diluted with PBS and plated onto Middlebrook 7H10 agar plates supplemented with OADC for enumeration of the CFUs. All the infection groups were analyzed in triplicates in each experiment, and total two independent experiments were conducted.