Evaluation of apoptosis by (A and B) flow cytometry after PI staining and (C) indirect immunofluorescence after TUNEL in untreated or FK-treated Nef transfectants cultured for up to 24 hours in 0.1% FCS. (A) Representative experiment shows the presence of apoptosis quantitatively evaluated by flow cytometry after PI staining in 24-hour serum-starved JH6.2, J.Bru2, and J.Bru.3 cells untreated (left) or treated with 10−5 mol/L FK (right). M1, apoptotic cells; x-axis, PI fluorescence (log scale); y-axis, relative number of cells. (B) Apoptosis was quantitatively evaluated by PI staining followed by flow cytometry at different culture times. Data are the mean ± SD of four-seven separate experiments in duplicate. (C) Representative of three separate experiments in which apoptosis was evaluated by TUNEL in confocal microscopy after 24 hours of serum starvation. Apoptotic cells were identified for the presence of intense fluorescent nuclei.
