Nheks

Human HaCaT cells, HDFs, NHEKs, NHEMs and murine B16F10 cells were purchased from Thermo Fisher Scientific (Waltham, MA, USA). HaCaT cells, HDFs and B16F10 cells were cultured in Dulbecco’s modified Eagle’s medium (HyClone, GE Healthcare Life Sciences, Logan, UT, USA) with 10% fetal bovine serum (HyClone) and 1% penicillin–streptomycin (LS202-02, Welgene, Daegu, Korea). NHEKs and NHEMs were cultured in keratinocyte media (MEP1500CA, Gibco, Life Technologies, Carlsbad, CA, USA) and Medium 254 (M254500, Gibco) supplemented with human keratinocyte and melanocyte growth supplement (S0015, S0025, Gibco) and 1% penicillin-streptomycin at 37 °C in a humidified mixture of air (95% (v/v)) and 5% CO₂. For LTAP exposure, 5 × 10⁵ cells were seeded in 35 mm dishes and incubated for 24 h. Before LTAP exposure, the medium was exchanged for 2 mL of new growth medium. The lid was opened, and the dish was placed 1 cm below the part where argon gas was ejected from the LTAP generator, and exposed for 1, 3, or 5 min. After exposure for the indicated time, the dishes were placed in a CO₂ incubator at 37 °C.

Primary neonatal human epidermal keratinocytes (NHEKs) was purchased from ThermoFisher Scientific. NHEKs were grown in serum free EpiLife medium supplemented with 0.06 mM CaCl₂, EpiLife Defined growth supplements (EDGS) (ThermoFisher Scientific) and antibiotics, and passage 3–5 cells were used for experiment. Cells at 60–80% confluence were starved overnight without EDGS prior to treatment. THP1 was purchased from American Type Culture Collection (ATCC) (Manassas, VA). THP1 was cultured in RPMI-1640 (Sigma) supplemented with 10% Hyclone fetal calf serum (Thermofisher Scientific), and antibiotics, and passage 3–5 cells were used for experiment. Cells at 60–80% confluence were differentiated by Phorbol 12-myristate 13-acetate (PMA, Sigma) for 24 hours and then starved overnight without Hyclone fetal calf serum prior to treatment.