Accuri c6 personal flow cytometer

Cells were harvested and labeled with anti-human glycophorin A (GPA)-PE (BD PharMingen, San Diego, CA, USA) antibodies at 4 °C for 20 min. To assess mitochondrial mass, cells were labeled with MitoTracker Deep Red (BD PharMingen) at 37 °C for 20 min. MMP was assessed by flow cytometry after labeling cells with cyanine dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbo-cyanine iodide, Thermo Fisher Scientific, Carlsbad, CA, USA). The cells were incubated with 10 μg/mL JC-1 for 20 min at 37 °C in 5% CO₂ and a humidified environment, washed twice with PBS containing 1% BSA and resuspended in 1% paraformaldehyde. Caspase-3 activity was measured using a CaspGLOW Fluorescein Active Caspase-3 Staining Kit (BioVision, Milpitas, CA, USA). Apoptosis was evaluated by labeling cells with Annexin V (BD Biosciences, San Diego, CA, USA) and propidium iodide (PI, Sigma). Stained cells were analyzed via flow cytometry using an Accuri C6 personal flow cytometer (BD Biosciences).
For immunofluorescence staining, cells were treated as previously described [16 (link)] with His-Tag (D3I1O) XP^® Rabbit mAb (Alexa Fluor^® 647 conjugate; Cell Signaling, Beverly, MA, USA), Alexa Fluor 633–phalloidin (Thermo Fisher Scientific, Waltham, MA, USA), GPA (BioLegend, San Diego, CA, USA), and 4,6-diamidine-2-phenylindole dihydrocloride (DAPI).

Brains were dissected from 40–50 3rd-instar larvae flies and incubated in 1 × Trypsin-EDTA (GIBCO) at room temperature with gently agitated using 200 μL micropipette tips every 15–20 min until no visible tissue fragments could be seen. The cell suspension was centrifuged at 800 g for 5 min, the pellet washed twice with PBS, and resuspended with serum-free Schneider’s Drosophila Medium for apoptosis assay. After staining, the cell suspension was analyzed by Flow Cytometry (Accuri C6 Personal Flow Cytometer, BD Biosciences, San Jose, CA, USA).
Cell apoptosis was assessed by flow cytometry following Propidium Iodide (PI) /FITC-Annexin V double staining assay. FITC-Annexin V negative cells together with Propidium Iodide stained nuclei were excluded and dead versus healthy cells assessed by FITC-Annexin V staining. The cells were incubated with H₂O₂ for 3 h, and then spun down and washed with cold PBS. The pellet was resuspended with 1 × binding buffer (FITC-Annexin V Apoptosis Detection Kit I, BD Pharmingen, Franklin Lakes, NJ, USA). The density of the cells was adjusted to 1 × 10⁶ per mL and 100 μL of cell suspension was transferred to a 1.5-mL Eppendorf tube. The cells were then loaded with 5 μL FITC-Annexin V and 10 μL PI for 15 min at RT in the dark, 400 μL of Binding Buffer was added to each tube, and the relative fluorescence was assessed by flow cytometry.

OSCC cell lines (SCC9, SCC15, SCC25, Ca9‐22, HSU3, TSCCA, Fadu) and the primary normal human oral keratinocytes (NHOK) cell lines were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China), and routinely cultured according to the recommendation and previously described.10 Cells were cultured in RPMI‐1640 medium (Gibco, Waltham, MA, USA) supplemented with 10% FBS (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin. All cells were cultured in a 5% CO₂ humidified atmosphere at 37°C. To propagate the CSC‐like fraction of the tumour cells, SCC15 and TSCCA cells were trypsinized and cultured in serum‐free stem cell medium containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 20 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor (EGF) (both from PeproTech, Rocky Hill, NJ, USA), 2% B‐27 supplement, 1 mM L‐glutamine and 1% P/S (all from Invitrogen, Carlsbad, CA, USA). CSC‐SCC15 and CSC‐TSCCA cells were washed in PBS and stained with the anti‐CD44‐FITC monoclonal antibody (BD Biosciences, San Jose, CA, USA). After 30 minutes of incubation in the dark in room temperature (RT) samples were analysed with the Accuri™ C6 personal flow cytometer (BD, Oxford, UK), and the data were assessed using the FlowJo software program (Tree Star, Ashland, OR, USA).