Supplementary Fig. 5: NLRP3 inflammasome in primary splenic DCs (relevant to Fig. 5).
From: Macrophages, rather than DCs, are responsible for inflammasome activity in the GM-CSF BMDC model
(a) Representative FACS plots showing the purity of the isolated primary splenic pDCs; cDC1, cDC2 from Fig. 5a,c. (b, d) FACS plots showing the gating strategy used to isolate primary splenic pDCs, cDC1, cDC2 (b) and moDCs or CD11c+ monocytes (d) for determining the data in panels c and e. (c, e) About 105 sorted splenic DCs from 3 mice were seeded into 96-well plates in triplicate for inflammasome activation assays and primed using CpG or LPS (100 ng/ml, 2–3 hours). Primed cells were stimulated for NLRP3 inflammasome activity by nigericin (10 μM). At 3 hours post-NLRP3 inflammasome stimulation, the cells were collected and (c) Inflammasome components were detected using IB (numbers on the right of each blot indicate the blot number: All images were cropped and their full-size images are presented in Supplementary Fig. 12). (e) Supernatants were collected after inflammasome stimulation, and IL-1β secretion was measured using commercial ELISA kits (symbols represent independent samples (n=3) and results are presented as mean ± sd). (f) FACS plots showing the comparison of in vivo GMtg “moDCs” with the ex-vivo BM-derived GM-DCs. Red boxes indicate a direct comparison between the pure populations. All FACS plots represent at least 3 independent biological experiments. (c), and (e) are representative from 2 independent experiments.
