Mouse macrophage colony stimulating factor m csf

NMR cells were used after 8 days of culture with 20 ng/ml mouse macrophage colony stimulating factor (M-CSF) (BioLegend) or immediately after cell sorting or one day of culture (NPM1 cells). Cells were cultured with pHrodo Green E. coli BioParticles Conjugate for Phagocytosis (Thermo Fisher Scientific) for 2 hours. Cultured cells were washed, fixed with 4% paraformaldehyde, and stained with the CD11b antibody and DAPI. Sorted cells and NPM1 cells were stained with 5 μg/ml Hoechst 33342 without fixation. Stained cells were observed by fluorescence microscopy. Image analyses were performed using ImageJ.

NMRs and mice were sacrificed humanely using isoflurane anaesthesia, and limbs were isolated. After removing muscles and cartilage tissue, the bones were crushed and suspended in PBS. The cell suspension was filtered through a 70 μm cell strainer (Falcon) and suspended in hypo-osmotic solution to remove red blood cells. The remaining cells after haemolysis were processed into a single cell suspension and cultured in RPMI-1640 (FUJIFILM WAKO) supplemented with 15% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, 0.1 mM non-essential amino acids, and 20 ng per mL mouse macrophage colony stimulating factor (M-CSF) (BioLegend) for 8 days^{64 (link)}. Dead cells were prepared by 200 J m⁻² of UVC irradiation to fibroblasts using a UV crosslinker (Analytic Jena). After UV irradiation, cells were cultured for 24 h, and dead cells were collected and stained using pHrodo (Thermo Fisher Scientific) according to the manufacturer’s protocol. The same amounts of pHrodo-labelled dead cells (5 × 10⁵ cells) were co-incubated with NMR or mouse bone marrow macrophage culture. After 2 h, phagocytosis was evaluated by measuring pH-sensitive fluorescence of pHrodo using the BZ-X image analyser (KEYENCE).