35 mm petri dish

A superficial skin biopsy around 2 × 5 mm in size was taken on the inside of the arm at the site of musculus biceps brachii. The procedure was performed under local anesthesia after informed consent. Tissue was cut into smaller pieces and put in culture in a 35-mm Petri dish (Corning, New York, NY, USA) under a coverslip according to the protocol described by Takashima et al. [45 (link)]. After a few weeks in a humidified 37 °C, 5% CO₂ incubator, fibroblasts started to grow along the edges of the tissue; during this time medium was changed 2–3 times per week (DMEM GlutaMAX^TM medium with 25 mM D-Glucose and 1 mM sodium pyruvate, supplemented with 13% fetal bovine serum (FBS), 10 mM HEPES and 1% antibiotic-antimycotic (10,000 U/mL penicillin, 10,000 mg/mL streptomycin, and 25 mg/mL amphotericin B), (Invitrogen, Carlsbad, CA, USA)). When cells were confluent, they were transferred to 25-cm² tissue flasks, passaged once to 75-cm² flasks, and then stored at −150 °C in FBS with 10% DMSO (Sigma-Aldrich, St. Louis, MO, United States).

A549 human lung cancer cells (CCL-185, ATCC) were plated in a T25 cell culture flask (Corning, NY) and grown in complete cell culture medium containing F-12K medium (30-2004, ATCC) and 10% fetal bovine serum (FBS, 30-2020, ATCC) in a cell culture incubator (37 °C, 5% CO₂). An appropriate amount of cell suspension solution was subcultured onto a 22 × 22 mm poly-L-lysine (PLL)-coated coverslip, housed in a 35-mm Petri dish (Corning, NY) and kept in the cell culture incubator for 1 h to allow the cells to attach to the coverslip. After that, 1.5 mL complete cell culture medium was added to immerse the coverslip. The cells were ready for use when the cell culture reached about 70% confluency. Two pieces of double-sided tape served as spacers were attached on top of a pre-cleaned glass slide. A coverslip with cells was placed on top of the tapes with two edges to form a chamber, with the cell side facing the glass slide. The two open sides of the chamber were sealed by nail polish to prevent evaporation. The sandwiched chamber was made right before imaging.

Both zygotes and parthenogenetic embryos were cultured in groups of 20–30 embryos per 50 μL droplet of KSOM_AA overlaid with embryo-tested mineral oil. Embryos were cultured in humidified atmospheres (Thermo) with 5% or 20% O₂. Culture plates (35-mm petri dish; Corning Inc., Corning, NY) were prepared and equilibrated in the incubator 1 h before embryos collection. All control groups were made in the same day and the same treatment groups were cultured in the same dish with distinct makers. In all experiments, embryos were cultured for 5 days in vitro, and observed respectively at 24, 72, 96 and 120 h in vitro and graded for the stage of development including blastula formation and hatch.

Alginate beads were generated using an electrostatic droplet generator (custom) operated on a 3% medium viscosity alginate solution (Sigma‐Aldrich, St. Louis, MO, https://www.sigmaaldrich.com) at 9,000 V over a bath of 100 mM BaCl² (Sigma‐Aldrich) solution. Bead size distribution was determined using a custom‐built MATLAB algorithm. White light images of the beads under ×5 magnification were displayed and the user defined each bead diameter by clicking on opposite bead edges. A total of 359 beads were imaged resulting in a size distribution of 161 ± 80 μm. The beads (2.5 ml sedimented) were rinsed and allowed to soak in 1 ml of high‐concentration (9.37 mg/ml) rat tail collagen I solution (Corning, Corning, NY, https://www.corning.com) for 6 days at 4°C. After soaking, 2.5 ml of beads were pipetted into a 35‐mm petri dish (Corning) and the excess collagen I was aspirated, and 8 ml of 2 mg/ml dopamine hydrochloride in 50 mM Tris buffer (Sigma‐Aldrich), pH8.5, was added. The dish was sealed with parafilm (Sigma‐Aldrich) and rotated at 16.5 rpm on a laboratory rotisserie (Thermo Fisher Scientific Life Sciences, Waltham, MA, https://www.thermofisher.com) for 1 hour at room temperature. Beads were then rinsed in the previously mentioned Tris buffer and then soaked in experimentally relevant, serum‐free media.

Both microglial and astroglial cells were sub-cultured by plating 5×10⁵ cells per 35 mm Petri dish (Corning) before RF exposure. Twenty four hours after seeding, the cells were divided into sham groups and exposure groups, and were subjected to RF exposure or sham exposure for 1, 3, 6, 12 and 24 hours. The cells were exposed to GSM 1800 MHz RF modulated by a rectangular pulse with a repetition frequency of 217 Hz at an average SAR of 2.0 W/kg with an intermittent duration of 5 min on and 10 min off. The computer randomly assigned the exposure waveguide and sham waveguide, achieving blind experiments, and then the cells received RF exposure or sham exposure simultaneously. There were no RF signals in the sham waveguide. After exposure, cells and cultured supernatants were harvested and subjected to subsequent tests. In addition, naive control groups and lipopolysaccharide (LPS) control groups were set up for each time-point. The naive controls did not receive any treatments and the LPS groups were treated with 1 µg/mL LPS. The STAT3 inhibitor Stattic (Santa Cruz Biotechnology, Texas, U.S.A.) pretreatments were carried out by administration of 20 µmol/L Stattic 45 min prior to 24-h RF exposure.

In order to prepare the plasma-treated solutions (PTS), 2.5 mL liquid was placed into a 35 mm petri dish (Corning; Merck, Darmstadt, Germany), together with a magnetic stir bar stirring at 300 rounds per min (rpm). If not otherwise noted, all of the liquids were treated at a frequency of 4 kHz or 8 kHz for 5 min; the corresponding control liquids remained untreated. The following liquids were used: Dulbecco’s modified Eagle’s medium (DMEM) (ThermoFisher Scientific, Schwerte, Germany) with 10% fetal bovine serum (FBS) (Anprotec, Bruckberg, Germany), 1% penicillin/streptomycin (P/S) and 1% L-glutamine (Sigma Aldrich GmbH, Steinheim, Germany) (abbreviation: DMEM + FBS), DMEM without any additives (abbreviation: DMEM–FBS), aqueous solutions of Tyrosine (Tyr) and Tryptophan (Trp) (1 mM in water; Sigma Aldrich GmbH, Steinheim, Germany), and melanocyte growth medium (MGM; CC-3249; Lonza Group Ltd, Basel, Switzerland) exclusively for the treatment of NHEM. The water used to prepare the solutions and the samples for the NMR and HPLC-TOFMS measurements was purified using a PURELAB Plus system (ELGA, LabWater, Celle, Germany).

The LUAD cells of each group in the logarithmic growth phase were inoculated on a 35mm petri dish (Corning, NY, USA) with 10 mL of 37°C pre-warmed culture medium and cultured for 2 weeks. Then, the petri dish was stained with crystal violet staining solution (Beyotime, Shanghai, China), and colonies with ≥10 cells were counted under the microscope with low magnification. After all preparations, three independent experiments were carried out.