Cd11b

Mice in the pre-immunized and acute (dpi 17) phases of EAE were euthanized, and their spinal cords were isolated and collected in ice-cold 1× Hank’s Balanced Saline Solution (HBSS). Spinal cord and brain cells were isolated by a density-gradient technique, as previously described [11 (link)]. Briefly, spinal cord and brain tissues were minced with a tissue homogenizer to obtain a single-cell suspension. Stock isotonic Percoll® (10-fold dilution in 10× HBSS without Ca²⁺ and Mg²⁺) was added to the cell suspension to form a 30% Percoll gradient. A 70% Percoll gradient was pipetted underneath the 30% Percoll cell solution and centrifuged at 800×g for 40 min. Then, the myelin layer was removed and the mononuclear cell interphase was isolated and resuspended in 1× HBSS. After blocking with an anti-mouse cluster of differentiation (CD)16/32 monoclonal antibody (Sony Biotechnology, San Jose, CA) for 10–15 min on ice, the cells were stained with phycoerythrin (PE)-Cy7 anti-mouse CD11b (BD Pharmingen™, San Jose, CA), fluorescein isothiocyanate (FITC) anti-mouse CD45 (Invitrogen, Rockford, IL), and allophycocyanin (APC) anti-mouse Ly6C (BioLegend, San Diego, CA) monoclonal antibodies. The cells were then sorted and analyzed using an SH800 Cell Sorter (Sony Corporation, Tokyo, Japan) by gating on CD11b⁺CD45^intLy6C⁻ for microglial cells.

Epididymal adipose tissues (VATs) of 16–20 weeks HFD-fed mice were mechanically chopped and then digested with collagenase II (Sigma-Aldrich, St. Louis, MO, USA; Cat. No. C2674) for 15 min at 37 °C. After passing cells through a 200 µm cell strainer (VWR, Cat. No. 100490-158) and centrifugation at 1000× g for 10 min, the pellet containing the stromal vascular cell (SVC) fraction was then incubated with red blood cell lysis buffer. SVC single cell suspensions were incubated with fluorescence-tagged antibodies against CD45 (Biolegend, Cat. No. 103116), CD11b (Biolegend, Cat. No. 101206), and F4/80 (Biolegend, Cat. No. 123116). CD45+CD11b+F4/80+ macrophages were purified using SONY MA900 flow cytometer (SONY). In addition, cells were stained with CD11c (Biolegend, Cat. No. 117343) and CD206 (Biolegend, Cat. No. 141715) antibodies to measure the levels of M1 and M2 activation. ATMs were then cultured in IMDM containing 10% exosome-free FBS to produce extracellular vesicles (EVs).