Isolation and Characterization of Adipocyte and Adipose Immune Cell Fractions

Adipocyte and adipose SVF, ART and ATM fractions were isolated from epididymal VAT10 (link). Briefly, adipocytes were incubated with biotinylated CD45 antibody (eBiosciences, 13-0451-85, 1:400 dilution) to remove leukocyte contamination and the purified CD45 negative adipocytes were used for the gene expression analysis and cell co-culture. ATMs and ARTs were isolated from SVF with biotinylated F4/80 (eBiosciences, 13-4801-85, 1:200 dilution) and CD3e (eBiosciences, 13-0031-85, 1:100 dilution) antibodies, respectively. BMDMs and PMs were generated39 (link). DCs were isolated from spleens of WT and aMHCII^−/− mice by using Miltenyi Pan Dendritic Cell Isolation Kit. Cell and tissue RNA samples were isolated and analysed, as previously described10 (link).