Fig. 1

Characterization of the NBM coculture system. a A schematic representation of NBM coculture system, showing formation of adherent and nonadherent BM compartments upon culture with patient serum. In this system, MM cell growth is measured with CD45/CD38 flow cytometry or direct bioluminescence of luciferase-expressing MM cells from day 1 to 7 of coculture. At these time points, conditioned medium and distinct cellular compartments including MM cells can be collected or purified for further analysis. b Prior to initiation of coculture, whole BM cells from healthy donors were cultured for 7 days in medium supplemented with MM patients’ serum; this was followed by 7 days without (top panel, NBM) or with (bottom panel, NBM + MM) MM cells. The whole BM was subjected to flow cytometry analysis of CD3 T cells, CD19 B cells, CD33 monocytes and CD56 NK cells. Percent values of each type of the cell in the NBM culture are shown. c Phase contrast images from coculture system (10X magnification): (upper left panel) multinucleated osteoclast-like cell (OC); (lower panel) cells that resemble macrophages (Mφ) and mesenchymal stem cells (MSC). Giemsa staining of nonadherent cells (upper right panel) from coculture system: typical hematopoietic cells such as lymphocytes (Lym) and monocytes (MO), and MM plasma cells (MM). d Phase contrast images (20X magnification) of adherent BM cells from coculture system: immunocytochemical staining of macrophages (Mφ) expressing CD68 (right)