Cells and culture
HeLa cells (human cervical carcinoma cell line obtained from Prof. M. Sekiguchi, Kyushu University) were cultured in Dulbecco’s Modified Eagle Medium (D-MEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA), 100 units/mL penicillin and 100 μg/mL streptomycin (Gibco, Thermo Fisher Scientific) at 37 °C in air containing 5% CO2 .
Preparation of PAL
Lactec (Ringer’s lactate solution, Na+ 130 mEq/L, K+ 4 mEq/L, Ca2+ 3 mEq/L, Cl− 109 mEq/L, L-lactate 28 mEq/L, pH 6.0 ~ 7.5, Otsuka Pharmaceutical Factory, Inc., Osaka, Japan; 3 mL) in a 35-mm tissue culture dish was irradiated using NEAPP [argon gas flow rate: 2.0 slm (L/min.), voltage: 9 kVp-p, frequency: 60 Hz; distance between the plasma source and the liquid surface: 6 mm] for 1 to 10 min. After irradiation, the PALs were stored at − 80 °C until use (at most 3 months). The frozen PALs were thawed at 4 °C for 3 h and kept at room temperature (26.3 ± 0.4 °C) for 1 h before use, then filtered through a Millipore Millex-GV 0.22 μm filter (Merck, Darmstadt, Germany). H2O2 was removed from PAL by adding 100 units/mL of catalase (Cat. from bovine liver, No. 035–12,903, Fujifilm Wako Pure Chemical Corp., Osaka, Japan) and the mixture was incubated for 10 min at room temperature (26.3 ± 0.4 °C). No hydrogen peroxide were detected (less than 0.3 μM) after this treatment.
Hydrogen peroxide concentration
The concentrations of H2O2 generated in PAL and diluted PAL were determined with the colorimetric method using an Amplex Red Hydrogen Peroxide Assay kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and by using a Digital Packtest hydrogen peroxide (DPM2-H2O2, Kyoritsu Chemical-check Lab., Corp., Yokohama, Japan) following the manufacturers’ protocols.
pH adjustment of PALs
The pH values of PAL and diluted PAL were measured with a pH meter (LAQUA, HORIBA, Ltd., Kyoto, Japan) and adjusted by adding 20% (vol/vol) of either 0.1 M phosphate buffer (pH 7.2) or 0.1 M citrate buffer (pH 6.1).
WST-8 assay for cell viability
The WST-8 assay (Cell Counting Kit-8, Dojindo Laboratories Co. Ltd., Kumamoto, Japan) was performed to determine the viability of the HeLa cells. The cells were harvested using trypsin-EDTA (Gibco, Thermo Fisher Scientific) and then seeded in 96-well plates (Falcon, Corning Inc., Glendale, AZ, USA). The plates were pretreated with 60 μL/well of 40.6 μg/mL collagen type I (Corning Inc.) for 30 min before use. The cells (5000 cells/100 μL) were added to each well, incubated for 24 h at 37 °C in a CO2 incubator, then treated with 100 μL of PAL for 120 min. After removing the test solution, the cells were cultured in 100 μL of fresh medium for 24 h, then 10 μL of WST-8 reagent was added to each well. After 2 h incubation at 37 °C in the dark, the absorbance at 450 nm was measured with a microplate reader (Multiskan GO, Thermo Fisher Scientific). Cell viability was calculated as follows: [(absorbance of samples)-(absorbance of background)] / [(absorbance of control) - (absorbance of background)] × 100%, where background represents the mixture of WST-8 reagent and D-MEM without cells.
Western blot analysis
Cells (2.0 × 106) were directly lysed in 100 μL of 2 × SDS sample buffer [62.5 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 25% (w/v) glycerol, 0.01% (w/v) bromophenol blue, 5% (v/v) 2-mercaptoethanol] (Bio- Rad, Hercules, CA, USA), incubated at 100 °C for 10 min and then chilled on ice. After sonication for 10 min (30 seconds on, 30 seconds off) the cell lysates (10 μL) were subjected to SDS-polyacrylamide gel electrophoresis and the separated proteins were transferred onto 0.2 μm pore size PVDF membranes (Bio-Rad). The membranes were blocked with 2% skimmed milk (Nacalai Tesque, Kyoto, Japan) in PBS containing 0.1% tween 20 (Nacalai Tesque), then reacted with anti-γH2AX (phosho S139) rabbit monoclonal antibody EP854(2)Y (Abcam, PLS Cambridge, UK) and anti-β-actin mouse monoclonal antibody AC-15 (Sigma-Aldrich). The membranes were further reacted with an appropriately diluted anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase (HRP) (GE Healthcare, Chicago, IL, USA), followed by reaction with Luminata™ Forte HRP substrate (Merck Millipore, Burlington, MA, USA). Luminescent signals were visualized using a ChemiDoc™ MP system (Bio-Rad).
Cells were cultured in D-MEM containing 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin and HAT supplement (0.1 mM hypoxanthine, 0.4 μM aminopterin and 16 μM thymidine) (Gibco, Thermo Fisher Scientific) for 2 weeks to eliminate hypoxanthine-guanine phosphoribosyltransferase (HPRT)-mutated cells from the cell population. The cells were then cultured in the same medium without aminopterin and 1.0 × 106 cells were treated with PAL or H2O2 for 2 h. The treated cells were cultured in normal medium to about 1 × 107 cells. Exponentially growing cells were divided into five 100-mm dishes at a density of 2 × 106 cells/dish and cultured in medium supplemented with 10 μM 6-thioguanine (Fujifilm Wako Pure Chemical Corporation) for 10 days to form colonies. To determine plating efficiency, 400 cells were inoculated onto a 100-mm dish, the formed colonies were fixed with 3.7% formaldehyde (Nacalai Tesque) in PBS, stained with 0.1% crystal violet (Fujifilm Wako Pure Chemical Corporation) and counted. Mutation frequency was calculated from the number of resistant colonies and the plating efficiency.
Cell cycle analysis
Cells were harvested with trypsin/EDTA and washed with PBS. Cell suspensions were passed through a cell strainer (Falcon, Corning Inc., Glendale, AZ, USA) and centrifuged. After removing the supernatants, cell pellets (1.0 × 106 cells) were suspended in 50 μL of PBS and the cells were fixed with 70% ethanol at − 30 °C. The fixed cells were passed through a cell strainer and centrifuged at 300×g for 5 min. After washing with PBS, the cells were suspended in 200 μL of Muse cell cycle reagent (Merck Millipore) and incubated at room temperature for 30 min in the dark. Cell cycle analyses were carried out following the recommended Muse Cell Analyzer (Merck Millipore) protocol.