Cd13 pe cy7

Unstimulated and IFNγ-stimulated AT-MSC were trypsinized and washed with FACS Flow (BD Biosciences, San Jose, CA). Cell suspensions were incubated with mouse-antihuman monoclonal antibodies against CD13-PE-Cy7; HLA-DR-PERCP; HLA-ABC-APC; CD31-FITC; CD73-PE; PD-L1-PE (all BD Biosciences); CD90-APC and CD105-FITC (R&D Systems, Abingdon, UK) at room temperature in the absence of light for 30 min. After two washes with FACS Flow, flow cytometric analysis was performed using FACSCANTO-II with FACSDIVA Software (BD Biosciences).

AT-MSC were isolated from subcutaneous adipose tissue of five healthy donors (2 females/3 males). The age of the donors was between 34-58 years old. The tissue was mechanically disrupted and enzymatically digested with 0.5 mg/ml collagenase type IV (Sigma-Aldrich, St. Louis, MO) in RPMI for 30 min at 37°C under continuous shaking. Thereafter, the cells were resuspended in MEM-α with 10% fetal bovine serum (FBS; Lonza, Verviers, Belgium), 2 mM L-glutamine and 1% P/S, filtered through a 100 µm cell strainer, and transferred to 175 cm² culture flasks (Greiner Bio-one, Essen, Germany). At 90% confluence AT-MSC (passage 2-6) were collected to generate MP. The phenotypic characterization of AT-MSC was performed by flow cytometry using FACSCANTO-II with FACSDIVA Software (BD Biosciences, San Jose, CA). AT-MSC were incubated with mouse-anti-human monoclonal antibodies against CD13-PE-Cy7; HLA-DR-PERCP; HLA-ABC-APC; CD31-FITC; CD73-PE; PD-L1-PE (all BD Biosciences); CD90-APC and CD105-FITC (R&D Systems, Abingdon, UK). All the antibodies were incubated with the cells for 30 min, at room temperature in the absence of light.

ASC were immunophenotypically characterized by staining for CD45-FITC, CD31-FITC, CD13-PECy7, CD73-PE, and CD90-APC (all BD Biosciences, San Jose, CA). For detection of IL-6 and IFN-γ receptors, 400,000 ASC (n = 3) were stained for two IL-6 receptor subunits (CD126 and CD130) and IFN-γ receptor (CD119). The cells were incubated with anti-CD126-PECy7 (BioLegend, San Diego, CA), anti-CD130-BV421 (BD Biosciences, San Jose, CA), anti-CD119-APC (SB Sino Biological Inc., Beijing, China), or isotype-matched control antibodies (eBioscience, San Diego, CA) in the dark for 30 min at room temperature. Thereafter, the cells were washed twice with FACSFlow (BD Biosciences) and measured on a FACS Canto II flow cytometer (BD Biosciences) and analyzed with Kaluza Analysis 1.3 software (Beckman-Coulter, Brea, CA).