EDTA-anticoagulated blood samples were drawn during morning hours. PBMCs were isolated by density gradient centrifugation on Fycoll-Paque® and cultured in RPMI medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (PAA), 2mML-glutamine (Gibco BRL), 100 U/mL penicillin (PAA) and 100 mg/mL streptomycin (Gibco BRL) at 37 °C in a humidified CO2 containing atmosphere. PBMCs were stimulated with Mtb H37Rv gamma-irradiated whole cells (10 μg/mL) and treated with DHEA or 7-OD (at 1 × 10−9M, 1 × 10−8M, 1 × 10−7M and 1 × 10−6M). We chose steroid concentrations that encompass the spectra ranging from sub-physiological to pharmacological levels, according to our previous work [17 (link)].
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