Anti hla dr pe

Following incubations, the plate was incubated in 4°C for 30 min and rubbed gently by pipette. Then, suspended monocytes were collected and transferred to FACS tubes. Then, cells were centrifuged and washed with 1 ml FACS buffer. Pelleted cells were surface stained with anti-CD14-BV421, anti-CD16-BV510, anti-CD86-PE-Cy7, anti-HLA-DR-PE, anti-CD206-APC, and anti-CD163-BV605 (BioLegend) for 30 min at 4°C. After washing with FACS buffer, cells were fixed by 4% paraformaldehyde (Sigma, UK) for 10 min at 4°C and then washed by FACS buffer. Finally, cells were resuspended in 250 μl FACS buffer and kept in 4°C until analysis. Data was acquired using an LSRII flow cytometer (BD Biosciences) configured with three lasers and 10 detectors and FACSDiva acquisition software (BD Biosciences). Compensation was performed using tubes of CompBeads (BD Biosciences) individually stained with each fluorophore and compensation matrices were calculated with Flowjo version 10 (TreeStar Inc., Ashland, OR, USA).

After pretreatment with TNF-α and IL-1β or the absence thereof, the hADSCs were harvested by digestion with 0.05% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA) (Specialty Media, Millipore, Billerica, MA, USA) and washed with PBS twice. The cell pellets were resuspended in PBS and incubated with anti-CD29-PE, anti-CD44-FITC, anti-CD34-FITC, anti-CD45-FITC, anti-CD80-PE, anti-CD86-PE, anti-HLA-ABC-FITC, and anti-HLA-DR-PE (all from BioLegend, San Diego, CA, USA) for about 30 min in the dark. The cytometry data were processed and analyzed using FlowJo software.

To characterize the expression of surface markers, the MSCs were immune-stained and detected by flow cytometry analysis. Antibodies used in this detection are anti-CD73-PE, anti-CD90-FITC, anti-CD105-PE, anti-CD14-FITC, anti-CD34-FITC, anti-CD19-PE, anti-CD45-APC and anti-HLA-DR-PE (Biolegend, San Diego, CA, USA).
To assess the apoptotic rates, H9c2 cells were seeded in a 6-well plate at 30,000 cells/well for 24 h and then desired treatments were performed for 24 h. Cells were stained using an Annexin V/PI staining kit (Beyotime, Shanghai, China) following the manufacturer’s instructions and detected by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).