Bv421 hamster anti mouse cd11c

After blocking with 2% rat serum, cells were stained for APC-Cy7 rat anti-mouse CD45 (BD), BV421 hamster anti-mouse CD11c (BD), PerCP-Cy5.5 rat anti-mouse I-A/I-E (BD), PE-Cy7 rat anti-mouse Ly-6C (BD), BV510 rat anti-mouse Ly-6G (BD), APC rat anti-mouse F4/80 (BD), PE anti-mouse/human CD11b (Biolegend), and Fixable Viability Stain 700 (BD). The stained cells were analyzed using BD FACS Canto II and DIVA software.

Mature adipocytes and stromal vascular fractions (SVF) were isolated as described below. Epididymal fat tissues were minced and incubated in a fresh digesting media of Krebs Ringer HEPES (KRH) buffer for 60 min at 37 °C and were separated into adipocytes and SVF by using mesh with a grid diameter 300 µm. The SVF cells were subjected flow cytometry analysis as previously described^{30 (link)}. The cells were first incubated at 4 °C for 10 min with Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block) (BD Pharmingen) to reduce nonspecific binding of antibodies to FcR receptors. The cells (1 × 10⁶) derived from epididymal fat tissues were incubated at 4 °C for 30 min in Stain Buffer (BD Pharmingen) with the relevant optimized amount of fluorochrome conjugated antibodies or the appropriate isotype controls: PerCP-Cy 5.5 Rat Anti-Mouse CD11b, BV421 Hamster Anti-Mouse CD11c, PE-Cy 7 Mouse Anti-Mouse CD45.2 (BD Pharmingen), Rat Anti-Mouse CD206 Alexa Fluor 647 (AbD Serotec), and Anti-Mouse F4/80 Antigen FITC (eBioscience). Dead cells were excluded from analysis using 7-aminoactinomycin D staining (BD pharmigen). All data were acquired with FACSAria I flow cytometer (BD Biosciences) and analysed using FlowJo software (TreeStar, Ashland, OR).