Trypan blue staining

Bone marrow cells from 8-week-old female C57BL/6 mice (SLC Japan, Shizuoka, Japan) were induced into macrophages in DMEM supplemented with 25 ng/mL macrophage colony-stimulating factor (M-CSF; R&D Systems, Minneapolis, MN, USA). BMMs were incubated with DMEM alone, 50 ng/mL mouse interferon-γ (IFN-γ; R&D Systems) and 10 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich), 20 ng/mL murine IL-4 (PeproTech, Cranbury, NJ, USA), or SHED-CM for 24 hours designated as M0, M1, M2 (IL-4), and M2 (SHED-CM), respectively. For cell mix experiments, M1 were mixed with M0 at a ratio of 1:1 and subsequently treated with SHED-CM for 24 hours (Fig. 2C). Their polarization states were examined by flow cytometric analysis and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR).
Postinduction, the medium was changed to a serum-free DMEM and incubated for next 24 hours, harvested, and centrifuged at 2000 × g for 5 minutes. The CM from M2 (SHED-CM) or M0 was designated as M2-CM or M0-CM. The protein concentration of each CM was adjusted to 3 µg/mL postharvesting the CM. Cell viabilities of the macrophages were evaluated with trypan blue staining (Fujifilm Wako).