Facscalibur software

The effects of polyphyllin VII on CRC cell apoptosis were determined using the Annexin V-FITC/PI apoptosis detection kit (EMD Millipore), according to the manufacturer's instructions. Briefly, cells were washed three times with PBS and resuspended in 1X binding buffer provided by the apoptosis kit. Subsequently, 2 µg/ml Annexin V-FITC and 2.5 µg/ml PI were added to the 1×10⁶ cells. Following incubation in the dark for 15 min, the early and late apoptosis statuses were analyzed using a flow cytometer, using FACSCalibur software (BD Biosciences). Apoptotic cells were subsequently analyzed via flow cytometry, using MoFlo XDP (Beckman Coulter, Inc.).

PDLSCs from P4 were used for these experiments. To characterize at a single-cell level, a multi-parametric flow-cytometry combining seven surface markers panel analysis was done. The antibodies CD105, CD73, CD166, CD90, CD34, CD45 and HLA-DR (BD biosciences, USA) were used to study the cell-surface characteristics. These markers were selected based on common MSC characteristics (Dominici et al., 2006 (link)). The combinations of CD34 FITC (Fluorescein isothiocyanate-fluorophore), CD 73 PE fluorophore, CD 34 FITC + CD73 PE, CD 45 FITC + CD 166 PE, HLADR FITC + CD 90 PE, CD 105 PE, for isotype control FITC (IgG1) + PE (IgG1), FITC (IgG2) + PE (IgG1) and one unstained tube was employed. This unstained tube served as negative control; and the isotype control tube had equal number of cells with corresponding antibody. Previously isolated and cryopreserved P4 BMMSCs served as positive control. The BMMSCs were obtained from the Biomedical Research Core Facility (BioRCF), Institute for Medical research (IMR), Kuala Lumpur, Malaysia. The steps were repeated for BMMSC. The PE conjugation was planned for all the expected positive markers and the FITC conjugation was planned for all the negative markers. The BD FACS Calibur software was used to analyse the marker expression.

The levels of HLA-A2 expression on PBMCs and HLA-DR, CD83, and CD86 expression on DCs were detected by flow cytometry performed in accordance with the instructions from BioLegend (San Diego, CA, USA). Briefly, 1×10⁶ cells/mL suspended in PBS were incubated with the following anti-human antibodies (2 µg/10⁶ cells): PE-HLA-A2, FITC-CD83, PE-CD86, and APC-HLA-DR (BioLegend). The cells were analyzed at room temperature in the dark for 30 min using the FACS Calibur software (Becton Dickinson, Franklin Lakes, NJ, USA). Fluorescein-conjugated isotype-matched unrelated antibodies were used as negative controls.