Pe cy 7 conjugated cd14

Approximately 100,000 freshly isolated SVF cells were stained for 30 min at 4 °C with surface antibody (Ab) solutions containing mixes of the following Abs: PE-conjugated CD1d, CD3, DC-SIGN, CD206 and CD16; FITC-conjugated HLA-DR, CD11c and CD45; APC-conjugated CD8 and CD163; PE-Cy-7-conjugated CD14, PB-conjugated CD4 and Pe-Cy5-conjugated CD206 (all Abs from BD biosciences, except Ab to CD163 and CD206, which were from Biolegend). When specified, approximately 400,000 SVF cells harvested after overnight incubation with brefeldin A were stained for 30 min at 4 °C for surface markers and intracellular cytokines using the BD intracellular cytokine fixation/permeabilization solution kit (BD Biosciences) according to the manufacturer’s instructions. The following Abs were used: PE-Cy7-conjugated CD14; APC-conjugated CD163; PE-conjugated IL-10, TNFα and IL-6 (all Abs from BD biosciences, except Ab to IL-6 which was from eBioscience). Exclusion of dead cells was performed in all experiments using the Dead Cell discrimination kit (Miltenyi Biotec, Germany). Cells were fixed with 1% paraformaldehyde and analysed with a LSR II flow cytometer using Diva 6 software (BD biosciences).

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.