Facs flow cytometer

T84 or LoVo cells with different treatment (1 × 10⁶ cells) were harvested, and resuspended with 100 μL binding buffer (KeyGEN BioTech, Jiangning, Nanjing, China) with ribonuclease (1 U/mL) and PI (propidium iodide, 0.5 µg) for 30 min. Cells were then incubated with 5 μL fluorescein isothiocyanate-conjugated annexin V, and then analyzed by FACS flow cytometer (Attune, Life Technologies, Darmstadt, Germany).

1 × 10⁴ cultured NSCLC cells per well of different treatment were harvested and washed with PBS. The cells were then fixed with 70% ethanol at 4°C for at least 30 minutes. After washing with PBS, ribonuclease (Abcam) was added to the cells, and the propidium iodide (200 µL, Abcam) was used to stain the cells. FACS flow cytometer (Attune, Life Technologies) was used to analyze the cell cycle.

The transfected TPC-1 and CAL62 were detached by trypsinization and then resuspended in the binding buffer of annexin V-FITC/PI double staining apoptosis detection kit (KeyGEN BioTech, Jiangning, Nanjing, China). Cells were then stained with 5 µL of PI and 5 µL of annexin V (KeyGEN BioTech). The apoptotic ratio was analyzed with a FACS flow cytometer (Life Technologies, Darmstadt, Germany).

FaDu cells post indicated treatment were harvested and suspended in the binding buffer of ApoDETECT Annexin V-FITC Kit (Thermo Fisher Scientific Inc.). After staining with fluorescently labeled Annexin V and propidium iodide (PI) (Thermo Fisher Scientific Inc.), the cells were performed with flow cytometry analyses by FACS flow cytometer (Attune, Life Technologies, Darmstadt, Germany) according to previous study [15 (link)]. For transwell assay, the FaDu cells post indicated treatment were suspended in a serum-free medium and plated into the upper chamber of the well (Corning, Tewksbury, MA, USA). Medium containing 15% fetal bovine serum was added into the lower chamber. Twenty-four hours later, cells in the lower chamber were stained with crystal violet and photographed under the microscope (Olympus). The upper chamber was precoated with Matrigel (BD Biosciences, Bedford, MA, USA) to investigate cell invasion according to previous study [15 (link)].

A498/R cells with indicated transfections were seeded in a 6-well plate and treated with or without 10 mM imatinib. Ten days later, the cells were fixed in methanol and stained with crystal violet before photographed under a light microscope (Olympus, Tokyo, Japan). For cell apoptosis analysis, A498/R cells were harvested, and then suspended in a binding buffer from ApoDETECT Annexin V-FITC Kit (Thermo Fisher Scientific Inc.). The cells were incubated with Annexin V-FITC and PI (Thermo Fisher Scientific Inc) and then the cell apoptosis was analyzed by FACS flow cytometer (Attune, Life Technologies, Darmstadt, Germany) [16 ].