Tcs sp microscope

The positive blue staining of β-galactosidase was used as a biomarker of cellular senescence. Cells were incubated with appropriate buffer solution containing X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) (Sigma-Aldrich) (Hernandez-Vallejo et al., 2013 (link)). The blue-stained cells observed at pH 6 and pH 4 were counted in eight fields at 20× magnification, and the percentage of pH6 positive blue senescent cells was calculated. We used the acidotropic dye LysoTracker (Invitrogen Corporation) to evaluate lysosomal mass. Cells were cultured in 96-well plates, washed and incubated with LysoTracker (50 nmol/L) in DMEM, for 30 min at 37 °C in the dark. Quantification was performed on a plate fluorescence reader (Spectrafluor Plus) at 620 nm. Autophagosomes density was visualized by immunofluorescence with an antibody directed against LC3II (LC3, M152-3, MBL international, Woburn MA, USA) in MSCs cultured with the HIV proteins for 20 days (Leica TCS SP microscope, 40× and 100× magnifications). Nuclear DNA was stained with DAPI.

PBMC adhesion assay was performed as previously described [20 (link)]. Briefly, fresh human PBMCs obtained from four different healthy blood donors were isolated by density gradient centrifugation (Ficollplaque plus, 17-1440-02, GE Healthcare Europe). PBMCs were counted and labeled for 30 min with fluorescent tracer calcein acetoxymethyl ester (10 μmol L⁻¹) (Sigma-Aldrich). Labeled PBMCs were added for 1 h at 37 °C to previously transduced HCAECs, cultured for 24 h in serum-free endothelial cell basal medium (Promocell). After washing with PBS, adherent PBMCs were counted in five different fields (Leica TCS SP microscope, 40× magnification). Results were normalized to HCAECs protein level for each condition.