Pacific blue conjugated cd4

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.

Flow cytometry assays were performed at LIM 60/DCIA/USP, as previously described (9 (link)). The monoclonal antibodies used in this study included phycoerythrin (PE)-Texas Red-conjugated CD3 (Invitrogen, Carlsbad, California, USA); Pacific blue-conjugated CD4, PE-Cy7-conjugated CD27, fluorescein isothiocyanate (FITC)-conjugated CD45RA, and Allophycocyanin (APC)-conjugated CD38 (all from BD Biosciences Pharmingen, San Diego, California, USA); PE-Cy5-conjugated CD127, Brilliant violet 785-conjugated CD28, Alexa Fluor 700-conjugated HLA-DR, PerCP-Cy5.5-conjugated CD57, and APC-Cy7-conjugated PD-1 (all from Biolegend, San Diego, California, USA); and PE-conjugated TIGIT (eBioscience, Carlsbad, California, USA). Figure S1 (refer to Appendix) shows the representative dot plots with strategies used to gate the different T-cell populations described in this study.