Streptomycin mixture

Pluripotent J1 (ATCC SCRC-1010) and R1 (ATCC SCRC-1011) mESCs were used as models in this study. The mESCs were cultured using sterile cell culture flasks (Corning, Netherlands) coated with 0.1% gelatin (Sigma, Germany), at 37 °C in a humidified atmosphere containing 5% carbon dioxide (CO₂). Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, Germany) supplemented with 15% ESC-qualified fetal bovine serum (Sigma, Germany), 0.1 mM MEM nonessential amino acids (Sigma, Germany), 0.1 mM 2-mercaptoethanol (Sigma, Germany), L glutamin (Sigma, Germany), 100 U/mL penicillin-100 microgram/mL (ug/mL) streptomycin mixture (Sigma, Germany), and 1000 units/mL of recombinant mouse leukemia inhibitory factor (LIF) was used for growing pluripotent mESCs. Cells were passaged every third or fourth day using trypsin EDTA, and the media were changed every other day.

A spontaneously immortalized human epidermal keratinocyte cell line (HaCaT; CLS Cell Lines Service GmbH, Eppelheim, Germany) was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Cytiva, Logan, Utah, United States, distributed by CliniSciences S.r.l., Guidonia Montecelio, Rome, Italy) with L-glutamine (4 mM) and a high glucose concentration (4500 mg/L), without sodium pyruvate, and supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning, Glendale, AZ, USA, distributed by Biosigma S.p.A., Cona, Venice, Italy) and 1% penicillin and streptomycin mixture (10,000 units/mL penicillin and 10 mg/mL streptomycin mixture, Sigma-Aldrich). The cells were grown in a humidified 5% CO₂ atmosphere at 37 °C. For all the experiments, HaCaT cells were seeded into 48-well plates (2 × 10⁵ cells/mL, 500 μL/well) or 96-well plates (1 × 10⁵ cells/mL, 100 μL/well) in a complete growth medium without antibiotics. The cells were PCR-tested for mycoplasma contamination every four weeks.