THP-1 cells were purchased from American Type Culture Collection (ATCC, Rockville, EUA) and cultivated with RPMI-1640 (LGC Biotecnologia, Brazil) supplemented with 2 mM L-Glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin and 10% heat-inactivated FBS (HyClone Laboratories, Canada) at 37°C, 5% CO2. Before infection, cells (5×105/well) were differentiated into macrophage-like cells (mTHP-1) using 80 nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) for 24 h. Then, mTHP-1 were washed with PBS (1×), which was replaced by fresh antibiotics-free medium. Subsequently, stimulation (3, 24, and 48 h) was performed with irradiated or live M. leprae (Multiplicity of Infection - MOI 10∶1, 100∶1) at 33°C. After infection, total RNA was extracted as described below.
PBMC from healthy donors were collected in K3EDTA-tube (Labor Import Com. Imp. Exp. Ltda, Brazil), and isolated by Ficoll-Hypaque density gradient. After centrifugation (2,500 rpm, 30 min, 25°C), the interface containing mononuclear cells monolayer was collected, washed twice with PBS 1×, and cultivated in RPMI 1640 (LGC Biotecnologia, Brazil) supplemented with 10% human A/B RH+ serum (Sigma-Aldrich).
PBMC from healthy donors were collected in K3EDTA-tube (Labor Import Com. Imp. Exp. Ltda, Brazil), and isolated by Ficoll-Hypaque density gradient. After centrifugation (2,500 rpm, 30 min, 25°C), the interface containing mononuclear cells monolayer was collected, washed twice with PBS 1×, and cultivated in RPMI 1640 (LGC Biotecnologia, Brazil) supplemented with 10% human A/B RH+ serum (Sigma-Aldrich).