PART 4. Effects of extremely low frequency electromagnetic fields on the tumor cell inhibition and the possible mechanism

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trypsin (optimal digestion time varied from cell to cell: 293 T, 1 min; Hepg2, 1 min; A549, 2 min). The digestion

was terminated in the medium, and all cells were added to the centrifuge tube. Then, the hole was cleaned with

normal saline many times and added to the centrifuge tube. At this time, the centrifuge tube contained all cells.

Whether cells were left in the hole was observed under the microscope.

Suspended cells: All cell suspensions were placed in a centrifuge tube. The holes were washed with normal

saline several times and placed in the centrifuge tube. At this time, the centrifuge tube contained all cells. The

pores were observed for cell residue under the microscope.

Counting: The cells were centrifuged, resuspended in an appropriate medium, diluted partially, and stained

with Trypan blue. Then, 10 µL of it was injected through the cover glass.

Calculation formula: Number of cells/4×volume×dilution ratio× 104 .

The cells in each group were counted three times for a total of three groups.

pH value detection . The membrane-permeable fluorescence dye BCECF AM and pH-sensitive fluores

cent probe 2',7'-bis-(2-carboxyethyl)-5-carboxyfluorescein (Beyotime Biotechnology, China) were used to assess

the intracellular pH value. The cells were cultured in 96-well plates and exposed to a 5-mT, 20-Hz magnetic field

for 2 h on the first 2 days. On day 3, the cells were resuspended in 100 µL of 25 µM BCECF AM in 0.04% Pluronic

F-127 working solution and incubated in a CO2 incubator at 37±0.18 °C for 1 h. The supernatant was replaced

with HHBS buffer (AAT Bioquest, USA), and the cells were subjected to magnetic field exposure for 2 h. The

supernatant was then replaced with PBS, and the pH values were determined using FITC flow cytometry (FACS

Calibur, BD).

Intracellular calcium concentration assays. The membrane-permeable fluorescence dye Calbryte

520 AM (AAT Bioquest) was used to assess the intracellular calcium concentration. The cells were cultured in

96-well plates and exposed to a 5-mT, 20-Hz magnetic field for 2 h on the first 2 days. On day 3, the cells were

resuspended in 100 µL of 5 µM Calbryte 520 AM in 0.04% Pluronic F-127 working solution and incubated in a

CO2 incubator at 37±0.18 °C for 1 h. The supernatant was replaced with HHBS buffer (AAT Bioquest), and the

cells were exposed to the magnetic field for 2 h. The supernatant was then replaced with PBS, and the intracel

lular calcium concentration values were determined using FITC flow cytometry (FACS Calibur).

Determination of membrane potential of cells. The membrane potential was determined using the

potential-sensitive fluorescence dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4) (3) (AAT Bio

quest). The fluorescent dye DiBAC4 (3) permeated into the depolarized cells with high membrane potential,

leading to an increase in the intracellular fluorescence intensity. DiBAC4 (3) was discharged from the hyperpo

larized cells, and the intracellular fluorescence intensity decreased. The cells were cultured in 96-well plates and

exposed to a 5-mT, 20-Hz magnetic field for 2 h on the first 2 days. The cells were cultured in 100 µL of HHBS.

On day 3, the cells were resuspended in 100 µL of 10 µM DIBAC4 (3) AM in 0.04% Pluronic F-127 working solu

tion and incubated in a CO2 incubator at 37±0.18 °C for 1 h. The supernatant was replaced with HHBS buffer

(AAT Bioquest), and the cells were exposed to the magnetic field for 2 h. The supernatant was then replaced with

PBS, and the cell membrane potentials were determined using FITC flow cytometry (FACS Calibur).

Sodium ion detection. The cells were cultured in 96-well plates and exposed to a 5-mT, 20-Hz magnetic

field for 2 h on the first 2 days. On day 3, the medium was replaced with 100 µL of HHBS and 100 µL of 10 µM

SBFI AM (Cayman Chemical) in a 0.04% Pluronic F-127 working solution. The supernatant was replaced with

HHBS buffer (AAT Bioquest) after 4-h incubation in a CO2 incubator at 37±0.18 °C, and the cells were exposed

to the magnetic field for 2 h. The sodium ion was determined at 330/80 excitation and 528/20 emission using a

microplate tester.

Potassium ion detection. The cells were cultured in 96-well plates and exposed to a 5-mT, 20-Hz mag

netic field for 2 h on the first 2 days. On day 3, the medium was replaced with 100 µL of HHBS and 100 µL of

10 µM PBFI AM (Cayman Chemical) in a 0.04% Pluronic F-127 working solution. After 4-h incubation in a CO2

incubator at 37±0.18 °C, the supernatant was replaced with HHBS buffer (AAT Bioquest), and the cells were

exposed to the magnetic field for 2 h. The potassium ion determination was conducted at 330/80 excitation and

528/20 emission using a microplate tester.

Trypan blue dyeing. 293 T, Hepg2, and A549 cells in the logarithmic growth phase were trypsin digested

and harvested. Raji cells were collected directly. Following centrifugation, the cells were stained with the vital

dye Trypan blue. The live cells were counted under an inverted light microscope (Carl Zeiss, Germany). The cell

growth inhibition rates were determined, and representative curves were plotted. Inhibition rate (%)=(num

ber of cells in the control group−number of cells in the magnetic field group) / number of cells in the control

group×100%.

Statistical analysis. All data were expressed as mean±standard error of the mean, and one-way analysis of

variance was used for comparison between multiple groups. The Student t test was used to compare the treated

versus untreated groups. A P value<0.05 indicated a statistically significant difference. The confirmatory results

at P<0.05 were obtained from repeating studies at least two times. Each set of experiments had three parallel

samples and each experiment was repeated three times.

Data availability

All data are presented in this published article and supplementary file.

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Acknowledgements

We thank Professor Peter K. Law from The Cell Therapy Institute, Wuhan, China, for technical assistance.

Author contributions

J.S. designed the study, performed most of the experiments, analysed data and wrote the manuscript. H.W.

advised on experiment design and helped with experiments. Y.J., X.J., Y.C and Y.Y.T. participated in study design

and performed experiments.W.Y.J. commented on the manuscript. Z.M, K.C, X.D.L, Z.L.C and J.M.W partici

pated in the production and measurement of magnetic field instruments. G.H.Y. designed and supervised the

study, interpreted data and wrote the manuscript.

Funding

The Special fund for cancer prevention and treatment of Shanghai Science and Technology Development Foun

dation (Grant number CT20200517A).

Competing interests

The authors declare no competing interests.

Additional information

Supplementary InformationThe online version contains supplementary material available at https://doi.org/

10.1038/s41598-023-34144-5.

Correspondence and requests for materials should be addressed to G.Y.

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