Facs flow cytometry analyzer

Cells were plated at a density of 9 × 10⁴ cells/mL in 6-well plates. When cells were grown to 80–90% confluence, cells were exposed to Tak treatment for 24 h. At the end of the treatment, the cells were serum starved for 3 h. After synchronization, the floating and attached cells were collected and centrifugated at 4 °C 1000× g for 5 min, and then cell pellets were washed twice with PBS and resuspended in 500 μL PBS with the addition of 4.5 mL 70% ethanol, followed by incubation at −20 °C for at least 2 h. Afterwards, the cells were collected and centrifugated at 1000× g for 5 min. Cell pellets were resuspended in 500 μL 1 mg/mL PI solution (0.1% Triton X-100, 100 μg/mL Rnase A, and 40 μg/mL PI) for 30 min on ice. A total of at least 10,000 cells per sample were recorded and monitored by a FACS flow cytometry analyzer (Beckman Coulter, Brea, CA, USA).

Cell proliferation was determined using MTT assay according to the manufacturer’s instructions. The absorbance was read at 450 nm on a multimode plate-reader (PerkinElmer, USA).
Cells in early and late apoptotic stages were quantified using an Annexin V-APC/PE double staining assay. Cells were collected and resuspended in 500 μL binding buffer at 1 × 10⁶ cells/ml, followed by staining with 5 μL Annexin V and 5 μL PE in the dark at room temperature for 15 min. Stained cells were immediately examined using a FACS flow cytometry analyzer (Beckman Coulter) with wavelength emission filters of 488–530 nm for the green fluorescence of Annexin V (FL1) and of 488–630 nm for the red fluorescence of PI (FL2).
For cell cycle assay, 3 × 10⁵ cells/well was seeded into a 6-well plate. After 24 h incubation, the cells were collected and fixed with 75% cold ethanol (1 mL PBS and 3 mL absolute ethanol) at −20 °C overnight. After that, the cells was incubated with 200 μL RNase A (1 mg/mL) and 500 μL propidium iodide (PI, 100 μg/mL) for 30 min at room temperature in the dark and analyzed using the FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The data were analyzed with ModFitLT V2.0 software (Becton Dickinson).
All experiments were performed for three independent times.

Cells in the early and late apoptotic stages were analyzed by an Annexin V-FITC/PI double staining assay. Early apoptotic cells showed the externalization of phosphatidylserine (PS) on the outer leaflets of the plasma membrane. Annexin V had a high affinity for PS. Cells were plated at a density of 9 × 10⁴ cells/mL in 6-well plates. When reaching confluence of 80–90%, cells were exposed to Tak or glutamate treatment for the indicated time, then both floating and attached cells were all collected and washed twice with PBS. Cells were resuspended in 500 μL binding buffer, followed by staining with 10 μL Annexin V and 5 μL propidium iodide (PI) in the dark at room temperature for 15 min. The stained cells were analyzed immediately using a FACS flow cytometry analyzer (Beckman Coulter, Indianapolis, IN, USA) with emission filters at wavelengths of 488–530 nm for green fluorescence of Annexin V (FL1) and 488–630 nm for red fluorescence of PI (FL2). A total of at least 10,000 cells per sample were acquired to ensure adequate data. The lower left quadrant indicates the normal cells (Annexin V−, PI−), the lower right quadrant indicates early apoptotic cells (Annexin V+, PI−), and the upper right quadrant indicates late apoptotic cells (Annexin V+, PI+).