Edta free trypsin

Cells were collected and seeded into 6-well plates. Cells were digested by EDTA-free trypsin (Shanghai Beyotime Biotechnology), stained with Annexin V-FITC and propidium iodide (Shanghai BestBio Science, Shanghai, China), and incubated in the dark for 15 min at room temperature. The cell cycle and apoptosis of each group were detected by an EPICS XL-MCL flow cytometer (Beckman Coulter, Brea, CA, USA) with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

Cells in the logarithmic phase were collected and incubated in 6-well plates at a density of 1×10⁵ cells/well. Cells were digested with EDTA-free trypsin (Beyotime Institute of Biotechnology), stained with Annexin V-fluorescein isothiocyanate and propidium iodide (MedChem Express LLC, Monmouth Junction, NJ, USA), and incubated in a dark place for 15 min at room temperature. The apoptosis rates of each group were detected using a flow cytometer (EPICS XL-MCL FCM; Beckman Coulter, Inc., Brea, CA, USA) with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. Data was analyzed using FCS Express version 3.0 (De Novo Software, Glendale, CA, USA).
Cells were treated for 2 h with the lysosomal inhibitors E64d and pepstatin A (10 mg/ml; MedChemExpress USA) or dimethyl sulfoxide (DMSO) following transfection. Monodansylcadaverine (MDC) powder was purchased from Sigma-Aldrich; Merck KGaA and dissolved in DMSO at 0.1 mol/l of the stock concentration. The working concentration was 50 µm/l. The cells were incubated with the MDC dye for 45 min in the dark at 4°C. The cells were subsequently washed with PBS three times. The positive rate of autophagy was determined using a flow cytometer (EPICS XL-MCL FCM; Beckman Coulter, Inc.). Data was analyzed using FCS Express version 3.0 (De Novo Software, Glendale, CA, USA).

The transfected cells were collected and seeded into 6-well plates (3×10⁵/well). The samples were subsequently digested in EDTA-free trypsin (Beyotime Institute of Biotechnology) and stained with 5 µl Annexin V-FITC and 5 µl PI (20%; Invitrogen; Thermo Fisher Scientific, Inc.) in the dark for 15 min at room temperature. The percentage of apoptotic cells, including early apoptosis and late apoptosis was measured by flow cytometry (BD Biosciences).

To perform the apoptosis assay, we utilized the Annexin V and PI Apoptosis Kit (US EVERBRIGHT, USA) as per the specified guidelines. Initially, SW1088 was treated with EDTA-free trypsin (beyotime, China) for digestion. The resulting cells were subsequently washed twice with pre-cooled PBS. Afterwards, the cells were resuspended in 1 × binding buffer and Annexin V and PI working solution were added sequentially to each centrifuge tube. After 15 min of incubation at room temperature, remove from the dark, and complete the detection on the flow cytometer as soon as possible after adding 400 μl of PBS.