Fig. 5.

MCSF-dependency of DCreg development. Co-cultures were established by overlay of Lin⁻ BM from C57BL/6J mice above a Transwell membrane to prevent contact of between hematopoietic progenitors and 5G3 stroma. Three replicates cultures were treated with the M-CSFR inhibitor GW2580 (10 mM), or left untreated (controls), with inhibitor replenished every 3 days at medium change. Treatment continued for 21 days, followed by a period of 14 days in the absence of inhibitor to assess recovery. Cell production was analysed flow cytometrically every 7 days through antibody staining to detect CD11b, CD11c and MHC-II expression and to delineate CD11c⁺CD11b⁺MHC-II⁺ cDC-like cells. (a) Gating procedure for identification of cDC-like cells produced in 14-day Transwell co-cultures in the presence of inhibitor and in 28-day co-cultures after inhibitor has been washed out. Controls contained no inhibitor. (b) Production of DCregs in Transwell co-cultures expressed in terms of number of cDC-like cells produced over time relative to cell number produced in 7-day control cultures. Data represent mean ± SE of three replicate co-cultures. Addition of GW2580 significantly inhibited cell production relative to control at each 7-day interval out to 28 days (p ≤ 0.05)