Fig. 4. Incubation of iDCs with KCl or soluble extracts from necrotic cells promotes surface expression of CD83. iDCs were treated for 4 hours as indicated and the percent positive CD83 cells was determined by flow cytometry. (A) Stimulation with 10 mM caffeine alone or 100 µM ATP + 1 ng/ml LPS did not significantly increase CD83 surface expression; incubation with 10 mM caffeine + 1 ng/ml LPS significantly increased CD83 surface expression (P<0.001). This effect was blocked by pretreatment with 20 µM dantrolene. (B) When KCl was added, cells were incubated with Krebs-Ringer saline (in which the NaCl had been substituted for KCl) for 30 minutes at 37°C; they were then centrifuged, and fresh medium containing 1 ng/ml LPS was added and cells were incubated at 37°C for 4 hours. (C) For experiments in which iDCs were incubated with the supernatant from necrotic HEK293 cells, extracts were prepared as described in the Materials and methods section and either used directly (+ necrotic cell extract ± 20 µM dantrolene) or dialysed overnight against 1xPBS (dialysed necrotic cell extract). Results are expressed as mean (± s.e.m.) percentage induction of CD83 surface expression of at least three experiments performed on iDCs purified from blood of different donors; values obtained by treating iDCs with 1 µg/ml LPS was considered 100%. ^*P and ^**P, significant difference in the treated population compared with iDCs treated with 1 ng/ml. ^***P, significant difference from iDCs treated with 1 ng/ml LPS + necrotic cell extracts.