Figure 3. In vitro generated Sirt6KO BMDCs show increased endocytic activity and impaired allostimulatory capacity. (A, B) WT and Sirt6KO BMDCs were harvested at day 7 and incubated with dextran-FITC for 30 min at 37°C or at 4°C. Thereafter, cells were stained for CD11c and finally analyzed by flow cytometry. (A) Gating was done on CD11c+ cells; one representative experiment out of six is presented. (B) Dextran-FITC+/CD11c+ Sirt6KO BMDCs were enumerated and their frequency was normalized to that of dextran-FITC+/CD11c+ WT BMDCs. Results are means ± SEM of six separate experiments, n=6 for each genotype. (C) purified allogeneic (BALB-c) CD4+ splenocytes (responders) were stained with CFSE and incubated with 5 μg/ml phythoemagglutinin (PHA) or with sorted, WT or Sirt6KO, CD11c+MHCII+ BMDCs at the indicated S:R ratios. Proliferation of alive (propidium-iodide negative) CD3+CD4+CD11c− cells was evaluated by carboxyfluorescein succinimidyl ester (CFSE) dilution after a 5-day incubation. One representative experiment out of three is presented, n=4 for each genotype.