Anti hla abc

Surface markers for hADSCs were evaluated using a mini Guava EasyCyte flow cytometer. 5x10⁴ cells were incubated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated antibodies, anti-CD105, anti-CD34, anti-STROI, anti-CD73, anti-CD45, anti-HLA-ABC, anti-HLA-DR (Invitrogen, Frederick, MD) for 30 min at 4°C in PBS and washed afterwards. Cell autofluorescence in channel F1 or F2 was subtracted to obtain a neat signal of each marker. 5x10⁴ cells were plated for differentiation tests. Adipogenic differentiation was performed in StemPro adipogenesis-conditioned medium (Invitrogen, Grand Island, NY) for one week, replacing medium every 3 days. Lipid droplets were stained by using red oil staining for validating differentiation. StemPro osteogenesis-conditioned medium (Invitrogen, Grand Island, NY) was used for osteogenic differentiation for 11 days, replacing media every 48 hours. Extracellular calcium deposits were identified by Von Kossa staining. hADSCs cultured in DMEN were also stained as controls.

Treated cells were characterized using the following monoclonal antibodies: anti-HLA-ABC (Invitrogen, Waltham, MA, USA), anti-PD-L1 (Becton Dickinson, Franklin Lakes, NJ, USA), and anti-HLA-DR (Becton Dickinson, Franklin Lakes, NJ, USA), conjugated with PE, Brilliant Violet 421. The staining was divided into two panels to avoid overlapping of emission spectra. Positive cells were distinguished from negative cells using unstained samples. FACS measurement was performed on the BD FACSCelesta Cell Analyzer.