Journal:

Article Title: Consequences of Fas-Mediated Human Dendritic Cell Apoptosis Induced by Measles Virus

doi:

Figure Lengend Snippet: Fas-dependent apoptosis of MV-infected DCs in MV DC-T cell cocultures. (A) At day 5, apoptosis was analyzed by FACS in cocultures of MV-infected DCs and syngeneic activated T cells. MHC-II-FITC–CD3-PE doubling staining confirmed the FSC/SSC gates for DCs and T cells. These gates were used to analyze the DiOC6-PI double staining in the presence of ZB4 blocking anti-Fas antibody or related isotype control. Apoptotic dead cells have decreased mitochondrial transmembrane potential and permeabilized membranes that render them DiOC6 negative and PI positive, respectively. In contrast, viable cells are DiOC6 positive and PI negative. Results are representative of three experiments; standard deviations were below 10%. (B) Immature DCs were not infected or MV infected and then cultured for 24 h. RNAs were extracted and used for RNase protection using the hAPO-3 probe kit and developed by a PhosphorImager system for 6 h. Local background has been subtracted from each signal. The highest value (1,000) was attributed to the highest signal; then the levels of mRNAs were quantified by densitometry and scanning comparison with control probes (GAPDH and L32). Data shown are representative of three experiments. (C) DCs were not infected (thick line) or MV infected (gray histogram), cultured for 3 days, stained with antibodies against Fas (UB2-FITC) or a control antibody, and analyzed by FACS. The expression of Fas protein on gated viable DCs is shown. Data shown are representative of three experiments; standard deviations were below 10%.

Article Snippet: In brief, RNA was hybridized overnight with the in vitro-translated 32 P-labeled probe (hAPO-3 kit; Pharmingen).

Techniques: Infection, Staining, Double Staining, Blocking Assay, Cell Culture, Expressing