Fig. 1: Expansion of the self-renewing macrophages in the long-term culture.

a The outline of the long-term culture with a repeated passage. BM bone marrow, MΦ macrophages. b Primary bone marrow-derived macrophages were detached, seeded at 3 × 10⁵ cells/well, and cultured for 4 days in the absence (none) or presence of 100 ng/ml rhM-CSF. The cells were serially passaged and the number of viable cells was counted (mean ± SD, n = 3). c After the fourth passage, cells were cultured for 2 weeks in the presence of rhM-CSF and stained by Wright-Giemsa staining. Colonies appeared are indicated by yellow arrowheads (left panel). d Colonies appeared as in c were collected, reseeded, and cultured for 7 days in the presence of rhM-CSF. e After the fourth passage, cells were cultured for 2 weeks in the presence of rhM-CSF (defined as “day 0”) and analyzed for the expression of MHC-II by flow cytometry. CD80 was added as a reference. The analysis was repeated as indicated. At day 12, cells were also analyzed for the expression of CCR2. The mean fluorescence intensity (MFI) and the percentage of positive cells (for MHC-II) are shown. f The MHC-II^low and MHC-II^high fraction observed as in e (day 12) were sorted and cultured for up to day 16 in the presence of rhM-CSF, and the number of viable cells was counted (mean ± SD, n = 3). g The MHC-II^low cells expanded in f were reseeded and cultured for 8 days in the presence of rhM-CSF. The cells were serially passaged and the number of viable cells was counted (mean ± SD, n = 3).