Trypsin without edta

After 48 h of transfection, the cells were detached with trypsin without EDTA (Thermo Fisher Scientific, Rockford, IL, USA) and collected in a flow tube. Following centrifugation at 2200 ×g for 30 m, the supernatant was discarded. The cells were washed with precooled PBS for 3 times, and centrifuged at 2200 ×g for 20 m, and the supernatant was discarded. According to Annexin-V-Fluorescein 5-isothiocyanate (FITC) apoptosis detection kit (Sigma-Aldrich Chemical Company), Annexin-V-FITC and propidium iodide (PI) (50: 1: 2) were mixed into Annexin-V-FITC/PI staining solution. Following the addition of 100 μL dye solution and incubation at room temperature for 15 m, the cells were added with 1 mL 2-[4-(2-Hydroxyethyl)−1-piperazinyl]ethanesulfonicacid buffer solution (Thermo Fisher Scientific). The apoptosis was detected by flow cytometry at 488 nm.

HMEC-1 cells were grown in 6-well plates at a density of 5 × 10⁵ and irradiated by different dosages of 590 nm LED. After 24 h, cells were isolated with 0.25% trypsin without EDTA (Gibco, Grand Island, NY, USA), centrifuged, resuspended with 4 °C PBS. Subsequently, irradiated cells were washed, stained with Annexin-V FITC & PI (BD Biosciences; San Jose, CA, USA), measured with a C6 flow cytometer (BD) and analyzed using FlowJo (BD). For reactive oxygen species (ROS) measurement, gathered cells were resuspended in serum-free medium containing DCFH-DA (1:1000, Biodragon immunotechnologies), kept at 37 °C for 20 min, washed and detected by flow cytometry.

Cells were digested with trypsin without EDTA (Gibco) and were washed twice with phosphate-buffered saline. The cells were treated with Annexin-V-FITC binding buffer, Annexin-V-FITC (Beyotime, China), and PI for 20 min at room temperature. Cell apoptosis was assessed by flow cytometric analysis using an FC500 flow cytometer (Beckman-Coulter). All experiments were performed in triplicate, and each data point represented the mean results of the triplicate experiments.

The Annexin V-FITC Apoptosis Detection Kit (Dojindo Laboratories, Japan) for CytoFLEX (Beckman Coulter, America) was used to distinguish viable cells, apoptotic cells, and necrosis cells. The specific process was as follows.
After the plasma-treated cells had been cultured for 24 h and 48 h, respectively, these cells (including the cell supernatant) were digested with trypsin (without EDTA) (Gibco, America) and collected. We had added 3 mL of PBS and centrifuged it at 1000 rpm for 3 min, then discarded the supernatant. The cell pellet was resuspended with 300 μL of binding buffer. 5 μL of Annexin V-FITC and 5 μL of PI solution were added to the cell suspension. And it was incubated for 15 min in the dark at room temperature. Finally, the results should be tested within 1 h.
Trypan blue is a regent which can differentiate the cell viability. The cells which can be stained to blue were dead. We use the Trypan Blue Staining Cell Viability Assay Kit (Beyotime, China) to calculate the number of viable cells after plasma treatment.

Annexin V-fluorescein isothiocyanate (FITC)/PI double staining was performed to quantitatively determine the percentage of cells undergoing apoptosis. Briefly, the NCI-H460 cells were seeded into a 6-well plates at 2×10⁵ cells/well and at 70–80% confluence, the cells were treated with 20, 40 or 80 µM luteolin or 300 nM Taxol at 37°C. Following treatment for 48 h at 37°C, the cellular monolayer was released using trypsin without EDTA (Gibco Life Technologies, Carlsbad, CA, USA). The cells were resuspended and incubated with annexin V-FITC (KeyGen Biotech Co., Ltd., Nanjing, China) for 15 min at room temperature, followed by PI staining. The cells were analyzed using a flow cytometer (Becton Dickinson, Mountain View, CA, USA). annexin V-FITC and PI double-negative cells were defined as normal cells, whereas annexin V-FITC-positive and PI-negative cells were defined as early apoptotic cells and annexin V-FITC and PI double-positive cells were defined as late apoptotic and necrotic cells. The annexin V-FITC-PI binding assay was determined at least three times. CellQuest software (BD Biosciences San Jose, CA, USA) was used to calculate the percentage distribution of normal, early apoptotic, late apoptotic and necrotic cells.