Screening Adipose Tissue Proteome

LEGENDScreen™ Human PE kit (Biolegend, Cat#700007) was used to screen the cell surface proteome. Fresh SVC from subcutaneous liposuction aspirates and PBMC from buffy coat blood were used in the screening as previously described (33 (link)). In brief, the tissue-derived and blood-derived cells were CD45 barcoded and stained with a backbone antibody panel allowing identification of the cells of interest. The cells were added to the plates provided in the LEGENDScreen™ kit and the staining was performed according to the manufacture protocol. Plates were run on an 18-color flow cytometer (LSR Fortessa, BD Biosciences) with 407, 488, 561 and 640 lasers using the BD FACSDiva™ Software (BD Biosciences). Flow cytometry data was analyzed using FlowJo v10 (Treestar, USA). Two manual gating approaches were performed to identify the NK cell subsets, a “broad” gating followed by a “fine” gating. Several adipose-enriched proteins identified from both gating approaches were validated for expression in additional adipose tissue samples, and a selection of these proteins was included in the 27-parameter flow cytometry experiment.