F4 80 af488

AT of male C57BL6 mice was cultivated using the tissue explant model as described above. After 72 h, different concentrations of palmitate were added to the cell culture medium. After 24 h cultivation with palmitate, the AT explants were digested using collagenase as described above. After the last centrifugation step, the cell pellet was resuspended in staining buffer and Fc-receptors blocked by anti-CD16/32 treatment (1:100, eBioscience, Frankfurt, Germany) for 10 min on ice. After centrifuging (300 g, 5 min, 4 °C, no break), the pellet was resuspended in staining buffer and stained with 1:100 dilution of F4/80-AF488 and CD11c-AF647 (BioLegend, San Diego, USA) for 20 min on ice. Cells were then centrifuged (300 g, 5 min 4 °C), resuspended in staining buffer and kept on ice until analysis. DAPI was added to exclude dead cells. CD11c-positive cells and mean fluorescence intensity of F4/80 positive, living, single cells were analyzed using a MACSQuant 16 analyzer.

Single-cell suspensions were obtained from individual mice and cell phenotypes were analyzed by flow cytometry as described previously (25 (link)) using the FACS Calibur machines (BD Biosciences). The gating strategy is displayed in Figure S1. The following labeled monoclonal antibodies were used: CD4-PErCp (dilution 1:300), CD4-AF488 (dilution 1:200), or CD4-PE (dilution 1:200) (all GK1.5, BioLegend), CXCR5-Fitc (L138D7, BioLegend, dilution 1:100), CD19-AF647 (6D5, BioLegend, dilution 1:400), B220-APC (RA3-6B2, BioLegend, dilution 1:400), F4/80-AF-488 (BM8, BioLegend, dilution 1:200), Ly6G-PE (1A8, BD Biosciences, dilution 1:200), CD44-FITC (IM7, BioLegend, dilution 1:150), CD62L-PE (MEL-14, BioLegend, dilution 1:200), CD11b-biotine (M1/70, dilution 1:100) with subsequent SAv-PerCP (BioLegend, dilution 1:500) staining. For intracellular staining of the lung cells, BD Fixation/Permeablization Kit (BD Biosciences) was used due to the recommendations. Briefly, after 16-17 hours of in vitro cultivation in the presence of mycobacterial antigens, cells were harvested, stained first for surface antigens, then fixed with Cytofix/cytoperm buffer for 30 minutes and then stained for intracellular cytokines with IFN-γ-PE (BD Biosciences dilution 1:150) and IL-17-PerCp (TC11-18H10.1, BioLegend, dilution 1:150) antibodies. Results are presented as mean ± SD.