Figure 3
HIV-‐1 proviral DNA and p24 protein detection in MSCs infected by HIV-‐1 strains. Analysis of HIV-1 proviral DNA by qualitative real time PCR (A): agarose gel electrophoresis of MSC infected by HIV-1IIIb and HIV-1ada at days 3 and 7 post-infection. Positive control was activated PBMC at day 3 and negative control was mock-infected MSCs. All experiments were performed using 5 × 105 MSC or activated PBMCs infected or not with HIV-1IIIb and HIV-1ada (5 ng/ml p24). Panel B shows DNA integrated proviral HIV-1. The total DNA extracted from 5 × 105 MSC or activated PBMC cells was run in agarose gel electrophoresis and, after the purification of cellular DNA as previously described (51), a nested Alu-PCR was performed. The MSCs challenged by HIV-1 strains were analyzed at day 7. Activated PBMCs infected with the two HIV-1 strains served as positive controls. A specific LTR 100 bp band is detectable in HIV-1 infected MSCs and positive controls. Panel C displays the cell supernatant p24 analysis. ELISA p24 kit was employed to analyze the p24 content in cell supernatant. This assay exhibits a sensitive limit at 3 pg/ml. The amount of p24 in MSCs challenged with HIV-1 strains was very low and, in these experimental conditions, slowly declined at later tested times. The positive controls were performed by activated PBMC infected with HIV-1 strains.