Af 700 conjugated cd3

Stromal vascular fraction (SVF) was isolated as previously described 28 and was used for flow cytometric analysis or the isolation of CD4⁺ T cells, IL‐6⁺ and IL‐6⁻CD4⁺ T cells. To isolate CD4⁺ T cells from SVF of two patients, cells were stained with Dead cell discriminator kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and with AF‐700‐conjugated CD3, Pacific Blue‐conjugated CD4, APC‐conjugated CD8, PE‐Cy‐7‐conjugated CD14 (all from BD Biosciences, Breda, The Netherlands). Using a FACS Aria CD3⁺CD4⁺CD14⁻CD8⁻ cells were sorted. Cells were plated in a 96‐well plate in DMEM 4.5 g/l glucose/F12/0.5% BSA/15 mM Hepes/glutamax/pen/strep and supernatant was harvested after 1 day of culture. Adipocytes were isolated as previously described 28 and used for co‐cultures with CD4⁺ T cells. PBMCs were isolated from buffy coats of healthy donors by standard ficoll plaque gradient. PBMCs were used for treatment with FCM or for isolation of CD4⁺ T cells. CD4⁺ T cells were purified using magnetic beads labelled with anti‐CD4 (Invitrogen Dynal, Oslo, Norway), followed by removal of the magnetic beads, according to the manufacturer's instructions. The purity of the isolated CD4⁺ T cells was typically above 95%. CD4⁺ T cells were used for co‐cultures with adipocytes.

Cultured cells (passage 1) were trypsinized and stained with a panel of antibodies for fluorescence-activated cell sorting (FACS) analysis. Approximately, 1 × 10⁵ cells were re-suspended in phosphate buffered saline (PBS) and incubated with IgG block for 5 minutes to block non-specific binding. The following antibodies were used: AF-700 conjugated CD3 (BD BioSciences, USA), PE conjugated CD14 (BD, Immunocytometry, USA), APC conjugated CD19 (BD BioSciences, USA), PE conjugated CD34 (BD, BioSciences, USA), APC conjugated CD44 (BD, Pharmingen, USA), FITC conjugated CD45 (BD Pharmingen, USA), PE conjugated CD73 (BD Pharmingen, USA), AF-700 conjugated CD90 (Biolegend, USA) and APC conjugated CD105 (Biolegend, USA). Cells were stained for 30 minutes at 4°C with the antibodies. After washing, samples were analyzed on a LSR II flow cytometer (BD, USA) and at least 10,000 events were acquired for each population. Data acquisition and analysis were performed using FACS DIVA software (BD Biosciences, USA). Unstained cells were used to establish flow cytometer settings. Debris and cells/particles with auto-fluorescence were removed by using a threshold on the forward scatter.