Pi for flow cytometry

To quantify the proportion of early-stage apoptotic cells the commercial kit “Vybrant Apoptosis Assay Kit #4” with YO-PRO-1 and PI for Flow Cytometry (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA; catalog number: V13243) was used. The cells were stained according to the supplemented manufacturer protocol. After 24 hours post-radiation, cells were collected and washed in cold phosphate-buffered saline (PBS). A total of 1 μL of YO-PRO-1 stock solution and 1 μL of PI stock solution were added to each 1 mL containing 1 × 10⁶ cells. Cells were incubated on ice for 20–30 min. Cells were analyzed by flow cytometry (BD FACSCalibur, Becton Dickinson, San Jose, CA, USA) using 488 nm excitation with green fluorescence emission for YO-PRO-1 (i.e., 530/30 bandpass) and red fluorescence emission for PI (i.e., 610/20 bandpass). A total of 50,000 events were acquired for each sample and analyzed with BD CellQuest Pro 5.1 software (Becton Dickinson, San Jose, CA, USA).

The effect of MFSD4 knockdown on induction of apoptosis was analyzed using the FITC Annexin V/Dead Cell Apoptosis Kit with FITC annexin V and PI for Flow Cytometry (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction. Briefly, control, siMFSD4-transfected and ultraviolet-light irradiated (60 s) MKN1 cells were washed in phosphate-buffered saline and centrifuged. Cells (5 × 10⁵ cells/ml) were incubated with 5 μL of FITC annexin V and 100 ng of propidium iodide and incubated at room temperature for 15 min. The cells were analyzed using a flow cytometer (BD FACSAria Fusion; BD Biosciences, Bedford, MA, USA).