Annexin 5 fitc

Cells were dyed with Annexin V‐FITC and propidium iodide (PI), and apoptosis was determined by flow cytometry (BD Biosciences, Franklin Lakes, NJ). Apoptosis was assayed through dual‐staining with Annexin V‐FITC and propidium iodide (PI; BD Biosciences, Franklin Lakes, NJ). In brief, transfected cells for 48 hr were incubated with Annexin V‐FITC and PI in dark, and subjected to analysis by flow cytometry (BD Biosciences, Franklin Lakes, NJ).

The SK-Hep1 cells were transfected with SAAL1 siRNA and the control siRNA, respectively for 48 h. Cells were detached by trypsin followed by fixation with 70% ethanol. The fixed cells were stained with FITC annexin V (BD Biosciences, CA, USA) for 30 min. FITC annexin V positive cells were analyzed using BD FACSCanto™ flow cytometry. Data were processed using FlowJo™ v10 software (v10, BD Biosciences, CA, USA).

Mouse blood cells were washed twice using Ringer’s solution supplemented with 5 mM calcium chloride. To detect FITC annexin V-positive cells, the erythrocytes were suspended in an annexin-binding buffer (BD Pharmingen, San Diego, United States) with FITC annexin V (1:200 dilution, BD Pharmingen, San Diego, United States) and incubated for 15 min at room temperature. Well mixing was performed by pipetting. Finally, erythrocytes were diluted five times in the annexin-binding buffer before analysis in the FACSVerse flow cytometer (Beckman Coulter, CytoFlex S, United States) at an excitation wavelength of 488 nm (blue laser) and emission wavelength of 530 nm.

Either stably transduced or parental BT-12 (1.25 × 10⁵ cells/well) and CHLA-266 (2.5 × 10⁵ cells/well) cells were seeded on 12-well plates. The next day, parental cells were either transfected using siPOOLs as described above or treated with 30 µM of CilioD dissolved in DMSO or DMSO as negative control. After 72 or 24 h, cells were harvested and propidium iodide (PI, Sigma‐Aldrich, #P4864) or Annexin V staining was performed. For PI staining, cells were fixed with ice cold EtOH (70%) for at least 30 min at 4 °C. Then, cell pellets were resuspended in 50 µl RNase A (0.1 mg/ml, AppliChem, #A3832), followed by the addition of 150 µl PI (50 µg/ml). Cells were stained overnight at 4 °C. For Annexin V staining, cells were stained with FITC Annexin V (BD Biosciences, San Jose, CA, USA, #556419 (FITC Annexin V), #556454 (Annexin V Binding Buffer)) and PI according to the manufacturer’s instructions. For both assays, cells were analyzed using the CytoFLEX from Beckman Coulter.

Cell cycle distribution by PI staining and apoptosis by AnnexinV-FITC (BD biosciences, San Diego, CA, USA)/PI staining were performed as previously described [62 ]. For analysis of CD133+ subpopulation, the cells were washed with PBS, and then incubated with antibody against CD133/1 conjugated with phycoerythrin (PE; MiltenyiBiotec, BergischGladbach, Germany) on ice in the dark for 20 min. After the cells had been washed with PBS, the CD133⁺ and CD133⁻ populations were analyzed by FACS. Active caspase-3 Apoptosis Kit (BD biosciences) was used to detect the heterodimer of 17 and 12 kDa subunits, which is derived from the pro-enzyme. For annexinV and caspase-3 double staining, cells were washed with 1× binding buffer and then incubated with FITC annexinV (BD biosciences) for 15 min at room temperature in the dark. After annexinV staining, caspase-3-PE staining was performed on the same cells following manufacturer instructions. ALDH activity was evaluated by ALDEFLUOR kit (ALDH, STEMCELL Tecnologies, Vancouver, BC, Canada) following manufacturer instructions. All flow cytometric analyses were performed by using BD Accuri™ C6 flow cytometer (BD biosciences).

Cell apoptosis analysis was carried out using FITC-Annexin V and propidium iodide (apoptosis detection kit, BD Biosciences, Franklin Lake, WI, USA) according to the manufacturer’s instructions. Briefly, cells were seeded in 10 cm Petri dishes and treated with cetuximab +/- cisplatin for 24 h or 48 h. Cells were harvested and counted, diluted in annexin buffer (BD Biosciences, Franklin Lake, WI, USA) at a concentration of 1 × 10⁶ cells per 100 µL, and stained with 10 µL of propidium iodide and 5 µL of FITC-Annexin V. After 15 min of incubation, cells were analyzed in flow cytometer on a BD LSRFortessa^TM (BD Biosciences, Franklin Lake, WI, USA) after satisfying QC using CST beads. Acquisition and data analyses have been performed using the BD FACSDiva^TM Software.