Facscalibur flow cytometer

For analysis of CD133 expression, cell-spheres were incubated with fluorescein isothiocyanate (APC)-conjugated anti-human CD133 antibody (eBioscience) according to the manufacturer’s protocol. After incubation for 1 h at 4 °C, the labeled cells were washed thrice with cold PBS and further analyzed using a FACS Calibur flow cytometer (BD Biosciences).
For analysis of CD44⁺CD24⁻ subset, cell-spheres were labeled with anti-CD44 and anti-CD24 antibodies and then incubate with anti-rabbit APC antibody and anti-mouse PE antibody (Bioss, China). CD44⁺CD24⁻ cell populations were gated and sorted out respectively by FACS Calibur flow cytometer (BD Biosciences).
For analysis of ALDH⁺ subpopulation, the ALDEFLUOR assay was performed according to manufacturer’s (STEMCELL Technologies) guidelines. Briefly, a single-cell suspension of BC cells was suspended in ALDEFLUOR buffer containing ALDH subtract and incubated at 37 °C for 30 min. A fraction of cells was incubated under identical condition in the presence of the ALDH inhibitor, diethylamino benzaldehyde (DEAB) to determine the background fluorescence. After the incubation, the cells were wash twice with wash buffer to remove excess ALDH substrate and inhibitors. Thereafter, ALDH⁺ subset was further analyzed using a FACS Calibur flow cytometer (BD Biosciences) according to the instrument’s manual.

CAL27 cells were treated with different drugs for the indicated times. The pretreatment was performed according to the manufacturer’s instructions for PI/RNase Staining Buffer (BD Biosciences, 550825). Then, the cells were analysed on a BD FACSCalibur^TM Flow Cytometer (Becton Dickinson, BD Biosciences, USA).
For the apoptosis experiment, the cells treated as described above were washed three times with PBS and centrifuged at 1000 rpm for 3 min. Then, the cells were resuspended in 1× binding buffer and fixed for 30 min. The cells were resuspended with Annexin V and PI dye in binding buffer at a ratio of 1:1:50, placed on ice for 15 min, and then tested on a BD FACSCalibur^TM Flow Cytometer (Becton Dickinson, BD Biosciences, USA).

For flow cytometric analysis of iNOS and eNOS expression, PDLSCs were harvested and fixed with 80% methanol and then permeabilized with 0.1% PBS-Tween for 20 min. Cells were then incubated in phosphate-buffered saline (PBS)/10% normal goat serum/0.3 M glycine to block nonspecific protein-protein interactions by the anti-iNOS and anti-eNOS antibodies (Abcam, 1 μg/1 × 10⁶ cells). Fluorescein isothiocyanate (FITC) goat anti-mouse immunoglobulin (Ig)G was used as a secondary antibody (BioLegend, 1 μg/1 × 10⁶ cells). Cells were analyzed with a fluorescein-activated cell sorter (FACS) Calibur flow cytometer (BD Immunocytometry Systems, San Jose, CA, USA).
For flow cytometric analysis of stem cell identification, PDLSCs were harvested and fixed with 80% methanol for 20 min. Fixed cells were incubated in sealing buffer for 30 min and then incubated in 3% bovine serum albumin (BSA)/PBS with anti-CD44, anti-CD45, and anti-CD146 antibodies (Abcam, 1 μg/1 × 10⁶ cells). FITC goat anti-rabbit IgG was used as a secondary antibody (BioLegend, 1 μg/1 × 10⁶ cell). Cells were analyzed with a FACS Calibur flow cytometer (BD Immunocytometry Systems).