Fig. 4

Functional change in iNOS+ MDSCs. a Total iNOS⁺ cells were harvested from the spleen of mice treated by VACV (10 d.p.i) (filled square) or untreated mice (open square). Isolated cells were mixed at the indicated ratios with HCT-116 target cells in a transwell system for 12 h. In addition, where indicated, 0.5 mM L-NIL was added to inhibit NO production. Cytotoxicity was measured by Alamar blue CTL assay and tumor killing was shown as % lysis. Experiments were performed in triplicate, n = 2. Error bars represent mean and SD. b In a separate experiment, HCT-116 cells isolated from the lower transwell chamber were analyzed by flow cytometry for apoptosis at the end of 12 h of culture period. The data is presented as % Annexin V⁺ cells. This is representative data out of two distinct experiments with similar results. c NO level was determined by nitrite measurement in the media. The culture supernatant obtained at 12 h of incubation period was used for the determination of nitrite level