Cd45 pe

The primary brown adipocytes were prepared in accordance methods with the previous publication [20 (link)]. Floating adipocytes were separated from the SVF (Stromal Vascular Fraction) by centrifugation at 300 ×g for 3 min. SVF was sequentially filtered through 70 μm filters before staining with the following antibodies for 10 min on ice: Sca-1-APC (Miltenyi Biotec, 130-093-223), CD11b-FITC (Miltenyi Biotec, 130-081-201), and CD45-PE (Miltenyi Biotec, 130-091-610). Following antibody incubation, cells were washed, centrifuged at 300 ×g for 10 min, and sorted with a BD FACS Aria (BD Biosciences, CA, USA). Data analysis was performed using BD FACS Diva software.

The cells were detached, dissociated into single cells using dispase (Sigma-Aldrich), and resuspended in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline PBS, 1% BSA; 5 × 10⁵ cells/100 μl FACS buffer). For cell-surface staining the cells were stained with primary antibodies CD73-PE (130-095-182, Miltenyi), CD105-FITC (130-094-926, Miltenyi), CD90- PE-VIO770 (130-099-295, Miltenyi), CD45-PE (130-098-141, Miltenyi), CD34-FITC (130-098-139, Miltenyi), CD24-FITC (130-098-861, Miltenyi), SSEA4-PE (FAB1435P, R&D), and TRA1-60-PE (09-0009, Stemgent) for 1 hour at 4 °C. For intracellular FACS staining, i.e. OCT4-A (130-105-606, Miltenyi) and OCT4-B (IC1759P, R&D), the cells were washed once in FACS buffer, fixed for 10 min in 0.01% paraformaldehyde (PFA), washed twice with PBS, resuspended in permeabilization buffer (PBS, 1% Triton) and stained as describe above. Cells were then washed twice with PBS, resuspended in FACS buffer and analysed by FACScalibur flow cytometry (CyAn ADP, Beckman Coulter). Collected data were analyzed with the Flowjo v.10 software package (Treestar, Ashland, OR, USA). Negative control was immunoglobulin G (IgG) primary antibody-specific isotypes.

The purity of isolated cells was analysed by flow cytometry on a BD Accuri C6 (BD Biosciences, Heidelberg, Germany). The 10⁶ cells were resuspended in 100 µL of MACs buffer and stained with 2 µL of anti-human CD14-FITC, 2 µL of CD45-PE, and 2 µL of propidium iodide solution (Miltenyi Biotech, Bergisch Gladbach, Germany) for 30 min in the dark at 4 °C. Conjugation with CD14-FITC antibody binds to classical monocytes which present the CD14 antigen, while conjugation with CD45-PE antibody binds to all human cells of hematopoietic origin, with the exception of erythrocytes and platelets. Propidium iodide was used to exclude the dead cells out of the cell analysis. The cells were centrifuged at 300× g for 10 min and resuspended in 500 µL of PBS for flow cytometric determination of purity. The analysis was carried out with BD Accuri C6 software (BD Biosciences, Heidelberg, Germany).

The differential immune cell count was determined by using either NovoCyte or Beckman Coulter CytoFLEX flow cytometers and analyzed with NovoExpress (Acea Biosciences, USA) or CytExpert software (Beckman Coulter, USA). Differential immune cell counts were performed as previously described (Chan et al. 2016 ). Leukocytes were identified as CD45⁺, alveolar macrophages as CD11c⁺Siglec-F⁺, eosinophils as CD11c⁻Siglec-F⁺, neutrophils as GR-1⁺CD11b⁺ or Ly-6G⁺CD11b⁺, B cells as CD3⁻/CD19⁺ and T cells as CD3⁺/CD19⁻ cells. All antibodies were from Miltenyi Biotech: CD45-VioBlue (130–110-802), CD45-PE (130–110-797), CD64-APC-Vio770 (130–118-685), Siglec-F-PE-Vio770 (130–112-334), Siglec-F-APC (130–102-241), CD11b-PE-Vio615 (130–113-807), CD11b-FITC (130–098-085), CD11c-PE-PerCP700 (130–110-842), CD11c-PE (130–102-799), Ly-6G-PerCP700 (130–117-500), Gr-1-PE (130–112-306), Gr-1-APC (130–102-833), CD3-APC-Vio770 (130–119-793), CD19-PE-Vio770 (130–111-885), Annexin-V-PE (130–118-363), and Viobility 405/520 Fixable Dye (130–109-814).