2.4. HIV-1 p24 antigen assay and cell viability test

The HIV-1 Bru strain was used in this study to infect CEM-SS cells which were derived from the human lymphoblastoid cell line CEM that expresses high levels of the CD4 antigen (^{Wain-Hobson et al., 1991}). The cells were cultured in the suspension medium RPMI-1640 (Gibco) containing 10% FCS. The CEM-SS cells were cultured at a density of 7.0 × 10⁴/well and infected with HIV_BRU at the multiplicity of infection (MOI) of 1. All solutions of the HS and shilajit samples were sterilized using 0.22 μm syringe-filters (Merck Millipore Ltd) and stored at −20 °C until use. CEM-SS cells were incubated for 24 h in the absence and presence of HS and SM samples in 96-well plates. The HS and shilajit samples were homogeneous and stable throughout the incubation period in a 96-well plate. As a control, we used the reference HIV-1 inhibitors: the reverse transcriptase inhibitor Azidothymidine (AZT) and the integrase inhibitor Raltegravir (RAL). They were purchased from Sigma-Aldrich. After 3 days following three washings, CEM-SS cells were cultured in 96 well plates for 3 days to measure p24 release. The level of virus replication was detected by ELISA using a «HIV-1 p24 antigen-ELISA-BEST » test system (Vector-Best, Novosibirsk, Russia). The effects on cell viability were monitored by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, which measures the reduction of yellow MTT to purple formazan by mitochondrial enzymes (^{Mosmann, 1983}).