Cd11b macs microbeads

Epididymal adipose tissue samples were digested using Collagenase Type II (1mg/mL) and cRPMI (RPMI, 1% pentastrep, 1% BSA), strained, and centrifuged. Following lysing, cells were resuspended and centrifuged twice in FACS buffer to create a single cell suspension. Cells were counted and the cell volume of each sample was computed prior to pooling samples into each of the four treatment groups. An isolation buffer (PBS supplemented with 0.5% BSA and 2 mM EDTA) was used to resuspend the cells and CD11b MACS microbeads (Miltenyi Biotec Inc., San Diego, CA, #130-049-601) were used according to manufacturer's instruction. Cells were separated by magnetic properties in the column matrix (MACS, Auburn, CA, #130-042-201) and then resuspended in flow buffer. CD11b positive cells were counted using trypan blue. CD11b cells were stained for F480⁺ (EBioscience, San Diego, CA, USA) and analyzed using a FC 500 (Beckman Coulter, Brea, CA) flow cytometer.