Srp3332

Macrophages were isolated from bone marrow of 6 weeks old SD rats as described in Ying et al. (Ying et al., 2013 ), plated on 8 well multichambered slides (Falcon 354108) at 100,000 cells per well in macrophage growth media consisting of IDMEM without HEPES (Sigma‐Aldrich I3390), with 10% heat‐inactivated foetal calf serum and 10 ng/ml colony‐stimulating factor (CSF) (Sigma‐Aldrich, # SRP3332) and fed every 2–3 days. After 7–10 days, growth media was replaced with M1 or M2 induction media containing either 100 ng/ml LPS (Sigma‐Aldrich, L2630) plus 50 ng/ml interferon‐gamma (Sigma‐Aldrich, I3257) or 10 ng/ml interleukin 4 (Sigma‐Aldrich, I3650) respectively without CSF, at normal pH or a pH of 6. Two days after transfer to M1 or M2 phenotype induction media, 1 μl of a central myelin enriched fraction (Lankford et al., 2017 (link)) was added to half of the wells to induce phagocytosis. DiR‐labeled MSC‐sEVs were added to wells three days after transfer to induction media. After 24 h, cultures were washed 3 times with plain IDMEM, fixed with 4% paraformaldehyde and stained with antibodies directed against CD206 and iNOS, visualized with fluorescent 488 and 594 wavelength secondary antibodies, counterstained with DAPI mounting media, examined, and photographed with a Nikon A1R multiphoton confocal microscope with NIS Elements software as above (Supplementary Table.1).