Compbeads set

To determine effects of chemotherapy and serological conditioning on HCAEC apoptosis, a panel of monoclonal antibodies (mAbs) were used. HCAECs were incubated for 30 min at 4°C with anti-CD31-FITC antibody (BioLegend, United States) and anti-Annexin V-PerCP-Cy5.5 antibody (BD Biosciences, United States). For subsequent intracellular staining, endothelial cells were incubated for 20 min at 4°C with 100 µL BD fixation and permeabilization solution (BD Biosciences, United States). HCAECs were then incubated for 30 min at 4°C with anti-cleaved caspase-3-V450 antibody (BD Biosciences, United States). Lastly, HCAECs were washed twice with 100 µL perm wash buffer (BD Biosciences, United States) and resuspended in 200 µL PBS (Invitrogen, Thermofisher, United States).
After performing calibration and fluorescence compensation with CS&T beads (BD Biosciences, United States) and compensation beads (BD CompBeads Set, BD Biosciences, United States), respectively, samples were analysed using the High Throughput System on a 12-colour flow cytometer (FACS Celesta, BD Biosciences, United States). Data were acquired using FACSDiva 6.0 Software (BD Bioscience, United States), with the sample acquisition stopping gate set to 10,000 events.

Co-expression of six epitopes, including the CD34, CD73, CD90, CD105, CD146, and CD166 was analyzed on trypsinized hOSSCs and reference cells using a batch of directly conjugated antibodies (all from BD Bioscience, Albertslund, Denmark) using the CytoFLEX and the Kaluza 1.3 software package (both from Beckman Coulter, Copenhagen, Denmark) as previously described.^{[21 ,22 ]} Compensation values were established for each run to control for the bleed-through utilizing the BD CompBeads Set (BD Bioscience) and the AutoComp Wizard in Summit 6.1 (Beckman Coulter), and the Kaluza 1.3 when analyzing the data. The gating protocol included a demarcation of each cell population from the cellular debris followed by the determination of the area of stable flow and selection of single cells. A cut-off value representing the top 2.5 percentile of the fluorescence minus one (FMO) control was then used to establish the positive population for each of the markers.^{[23 ,24 ]}