Amphotericin b solution

HEK293T were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 4 mM L-Glutamine (Gibco) and 80 mg/mL gentamicin (Laboratorios Normon, Madrid, Spain), 100 U/mL penicillin/streptomycin (Life Technologies, Grand Island, NY, USA) and 1% amphotericin B solution (Lonza, Hopkinton, MA, USA) in 5% CO₂ at 37 °C conditions. The immortalized human neural stem cell line derived from ventral mesencephalon of fetal brain (ReNcell VM) was purchased from EMD Millipore (Billerica, MA, USA). ReNcells VM were plated onto Corning Matrigel hESC-Qualified Matrix (Corning, Bedford, MA, USA)-coated T75 cell culture flasks (BD Biosciences, San Jose, CA, USA) and maintained in neurobasal medium (Gibco, Thermo Fisher Scientific, Grand Island, NY, USA) supplemented with 2% (v/v) B27 supplement (Gibco), 20 ng/mL recombinant human epidermal growth factor (Peprotech, NJ, USA), 20 ng/mL recombinant human basic fibroblast growth factor (Peprotech, NJ, USA), 100 U/mL penicillin/streptomycin (Life Technologies, Grand Island, NY, USA) and 1% amphotericin B solution (Lonza, Hopkinton) in 5% CO₂ at 37 °C conditions.

Validated U–373 MG, U–87 MG, and MDA–MB–231 cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 4 mM L–glutamine (Gibco ID 25030081, Waltham, MA, USA), 80 mg/mL gentamicin (Normon Laboratories, Tres Cantos, Spain), and 1% amphotericin B solution (Lonza ID 17–836E, Basel, Switzerland), in 5% CO₂ at 37 °C. Sulforaphane (SFN) was purchased from LKT Laboratories (ID S8044, St Paul, MN, USA). Paraquat (PQ) was purchased from Sigma Aldrich (ID M2254, St. Louis, MO, USA). For synchronization at G1/G0, cells were FBS–deprived for 64 h and then replated in a standard medium (0 h point). For synchronization before S entry, a double thymidine pulse was performed. Cells were treated with 2.5 mM thymidine (Abcam, ID ab143719, Cambridge, UK), and then washed with PBS twice and maintained in thymidine–free medium for 8 h. Then, a second thymidine pulse was performed under the same conditions, and cells were finally chased to standard medium (0 h point). For synchronization in the M phase, cells were treated with 75 ng/mL nocodazole (Sigma Aldrich, ID M1404) for 16 h. Then, round and partially detached cells were harvested and plated in a nocodazole–free medium (0 h point).

Freshly excised full thickness human skin was incubated in Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (Merck, Darmstadt, Germany), and 1% penicillin/streptomycin + 1% amphotericin B solution (Lonza, Verviers, Belgium) at 37 °C in a humidified atmosphere of 5% CO2. Skin fragments were treated with 1% enhancer in 60% PG, 20 µl/cm², 60% PG alone or left untreated for 24 or 48 h. Then the skin was washed rinsed with distilled water and gently blotted dry. Skin viability was evaluated using the 2,3,5-triphenyltetrazolium chloride (TTC) reduction test^{57 (link)}. Punch biopsies of 5 mm diameter were incubated in 1 ml of 1.5% TTC and 3% sodium succinate in phosphate buffered saline at pH 7.4 at 37 °C under nitrogen in the dark. After 1 h, the skin samples were dried and the dark red formazan derivative produced by the mitochondrial enzymes was extracted with 1 ml of 2-methoxyethanol for 12 h. Absorbance was measured at 490 nm using Tecan Infinite 200 M plate reader (Tecan, Grödig, Austria) and corrected using skin samples devitalized by boiling as a negative control. The viability of skin exposed to the different preparations was expressed as a percentage of untreated control skin.