2.1 Cell culture
The human normal epithelial prostate cell line PNT1A and prostate cancer cell line DU145 were obtained from Prof. YongQuan Chen’s lab at Jiangnan University which were purchased from Chinese Academy of Sciences Cell Library. All cell lines were cultured following supplier’s recommendation. Cells were grown at 37 °C and 5% (v/v) CO2 in a humidified incubator.
2.2. Plasma source
The helium plasma generator is composed of a plasma jet gun, an iron stand, an oscilloscope, a rotor flow meter, and a helium gas bottle. We applied a voltage of 1.2 KV on the high-voltage electrode, the gas flow rate was 1.2 L/min, the distance between the plasma jet gun and the surface of the culture medium was 1 cm, and the processing time was 2-5 min.
2.3. Cell viability assay
Prostate cancer cells were plated in 96-well plates at a density of 1×104 cells/well in 100 μL of complete culture medium followed by CAP treatment for 5 min. The first measurement was taken 8 h post CAP exposure. Cell viability was assessed by Cell Counting Kit-8 (Dojindo, Japan) according to the manufacturer’s protocol. The absorbance was measured at 450 nm using EZ Read 800 microplate reader (Biochrom, UK).
2.4. Transwell assay
Cell medium was added on the lower layer of 24-well culture plate and the chambers were placed in the medium. Cells with and without CAP exposure were incubated for 72 h followed by pancreatic digestion and re-suspension. Cells were added to the chambers, with 2×105 cells per well. The culture media inside the chambers were discarded after 20 h, and cells were washed by phosphate buffered saline (PBS). Migrated cells under the chambers were fixed by methanol followed by staining using 0.1% crystal violet solution.
2.5. Wound-healing assay
Cells were plated in 6-well plates at a density of 1×106 cells/well, and scratched by a p100 tip when cells attached to the bottom of the plate as a monolayer and reached the confluence of 90%. Cells in each well were incubated using CAP-activated serum-free medium for 24 h, 48h and 72h. Scratches were visualized using inverted phase contrast microscope and images were captured using a digital microscope camera. Images were captured for each well immediately after the medium was replaced with PAM, and after 8 h, 16 h and 24 h of incubation. The Image J software was used to quantify the cell migration area.
2.6. Cell apoptosis assay
Apoptosis ratio was quantified using annexin V-FITC apoptosis detection kit (Dojindo, Japan). Cells were attached to a 6-well plate at a density of 1×105 cells/well, and the medium was replaced by 2 mL PAM and incubated for 24 h when cell density reached 80% confluence. Cells were harvested and stained using annexin V and propidium iodide (PI). FACSCalibur (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA) was used to calculate the apoptosis rate. Results were analyzed using FlowJo software (Ashland, OR).
2.7. Cell cycle assay
Cells were grown in 6-well plates, PBS-washed and digested using EDTA-free trypsin. Cells were centrifuged at 1000 rpm/min for 5 min, and supernatant was removed to collect the pellets. Cell pellets were washed using 500 μl PBS, centrifuged at 1000 rpm/min for 5 min, and the supernatant was removed to retain the pellets. Cell pellets were re-suspended in 70% ethanol and placed in a 4 ° C refrigerator overnight. Fixed cells were centrifuged at 1000 rpm/min for 5 min to remove the supernatant, and cell pellets were suspended in 500 μl PBS, supplemented with 5 μl Propidium iodide staining agent (purchased from Beyotime), and mixed on ice for 30 min. Cell cycle detection was performed using a BD Accuri C6 flow cytometer, and data analysis was performed using Flowjo software.
2.8. Intracellular hydrogen peroxide determination assay
ROS fluorescent probe-DHE (dihydroethidium, sigma, United States) was used to measure intracellular superoxide anion that reflects the level of ROS in general. Data acquisition was performed under Nikon’s fluorescence microscope following manufacture’s protocol.
2.9. Western blot
The 6-well plates of the cultured cells were taken out, washed with PBS at 4 °C, and then lysed with RIPA protein lysate containing protease inhibitors and phosphatase inhibitors for 5 min on ice, followed by centrifugation at 12,000 rpm for 20 min at 4 °C. The supernatant was taken and the protein concentration was determined using a BCA protein assay kit. The same amount of protein (40 μg) was added to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to the PVDF membrane. Non-specific binding sites were blocked for 1 h at the room temperature (RT) using 5% skim milk, followed by the PVDF membrane wash for 15 min, addition of primary antibody for 12 h and an appropriate horseradish peroxidase conjugated secondary antibody at RT for 1 h. Visualization of reactive protein bands was performed using High-sig ECL (enhanced chemiluminescence) western blotting substrate (Tanon), and the expression level of reactive protein bands was quantified using Image J software.