Mojosort mouse cd45 nanobeads

Stromal vascular cells (SVCs) were collected from adipose tissues as indicated by Muir et al. (3 (link)). After cardiac perfusion, adipose tissues were collected, minced finely to 3–5 mm pieces, and added to ice-cold HBSS plus Ca/Mg. Up to 1.5 g of tissue per sample was digested in 10 mL of 1 mg/mL collagenase II (Sigma; catalog C68850) in HBSS plus Ca/Mg at 37°C for 45 minutes with vigorous shaking. Digests were filtered through buffer-soaked 100 micron cell strainers and centrifuged at 300g at 4°C to pellet SVCs. SVCs were enriched for CD45⁺ immune cells using MojoSort Mouse CD45 Nanobeads (Biolegend; catalog 480027) following the manufacturer’s protocol. Briefly, SVC pellets were resuspended in 1 mL of MojoSort Buffer, pooling the 4 samples from each cohort (ND group, and 8-week and 14-week HFD groups) into a single, respective cohort tube, then filtered through a 70 μm cell strainer and placed in 5 mL polypropylene tubes. After addition of nanobeads, samples were sequentially processed for magnetic separation. Three magnetic separations in total were performed on the labeled fractions for increased purity. Final cell suspensions were filtered through 40 μm pipette tip filters. Cell viability was >80% with <15% aggregation.

Stromal vascular cells (SVCs) were collected from adipose tissues as indicated by Muir et al. (3 (link)). After cardiac perfusion, adipose tissues were collected, minced finely to 3–5 mm pieces, and added to ice-cold HBSS plus Ca/Mg. Up to 1.5 g of tissue per sample was digested in 10 mL of 1 mg/mL collagenase II (Sigma; catalog C68850) in HBSS plus Ca/Mg at 37°C for 45 minutes with vigorous shaking. Digests were filtered through buffer-soaked 100 micron cell strainers and centrifuged at 300g at 4°C to pellet SVCs. SVCs were enriched for CD45⁺ immune cells using MojoSort Mouse CD45 Nanobeads (Biolegend; catalog 480027) following the manufacturer’s protocol. Briefly, SVC pellets were resuspended in 1 mL of MojoSort Buffer, pooling the 4 samples from each cohort (ND group, and 8-week and 14-week HFD groups) into a single, respective cohort tube, then filtered through a 70 μm cell strainer and placed in 5 mL polypropylene tubes. After addition of nanobeads, samples were sequentially processed for magnetic separation. Three magnetic separations in total were performed on the labeled fractions for increased purity. Final cell suspensions were filtered through 40 μm pipette tip filters. Cell viability was >80% with <15% aggregation.

Murine TNF-α and M-CSF were purchased from Peprotech. Recombinant mouse IFN-β was from PBL Assay Science. The control IgG (rabbit) was obtained from Santa Cruz Biotechnology, and IFN-β neutralizing antibody (rabbit polyclonal antibody against mouse interferon-beta) was from PBL Assay Science. Mouse IFN Alpha All Subtype ELISA Kit and VeriKine-HS Mouse IFN Beta ELISA Kit were purchased from PBL Assay Science. MojoSort Mouse CD45 Nanobeads were obtained from Biolegend.