Penicillin streptomycin amphotericin b solution

Immortalized human keratinocytes (HaCaT) cells were grown at 37 °C temperature in a humidified atmosphere consisting of 95% O₂ and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM, low glucose, Gibco, Carlsbad, CA # 11885092) that was supplemented with 10% FBS (Gibco, Carlsbad, CA), 100 IU/ml penicillin, and 0.1 mg/ml streptomycin^{37 (link),95 (link),96 (link)}.
Primary Human Epidermal Keratinocytes (HEKa) cells (ATCC, Manassas, VA #PCS-200-011) were cultured in dermal cell basal media (ATCC, Manassas, VA #PCS-200-030) supplemented with human keratinocyte growth supplement (ATCC, Manassas, VA #PCS-200-040) and Penicillin–Streptomycin–Amphotericin B solution (ATCC, Manassas, VA #PCS-999-002) and incubated at 37 °C in a CO₂ incubator. After every 48 h, the media was changed until the cells reached 80–90% confluency. HEKa cells would start to become senescent at about passage 3 or 4. Therefore, all the experiments were performed with cells at passages 2 to prevent cell senescence.

Primary bladder epithelial cells (PCS-420-032, ATCC, Manassas, VA, USA) were purchased and expanded using the provided protocol. Briefly, cells were transferred to a T75 flask at 4000 cells per cm² with Prostate Epithelial Basal Medium (ATCC, Manassas, VA, USA) supplemented with Corneal Epithelial Cell Growth Kit (ATCC, Manassas, VA, USA) and Penicillin–Streptomycin–Amphotericin B Solution (ATCC, Manassas, VA, USA) at 5 mL of complete growth media per 25 cm². Every 48 h, spent medium was removed and fresh medium was added until approximately 80% confluence was reached. Upon reaching approximately 80% confluence, spent medium was removed and cell layer was detached using 0.05% Trypsin–EDTA (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Trypsin–EDTA was neutralized with equal volume fetal bovine serum (Corning, Corning, NY, USA). Cells were collected and centrifuged (Sorvall ST8, Thermo Fisher Scientific, Waltham, MA, USA) for 5 min at 150 g. Cells were counted and either frozen in working volumes with 10% dimethyl sulfoxide (Thermo Fisher Scientific, Waltham, MA, USA) or used for wound-healing assay at passage 4.

Normal human dermal fibroblasts were kept in Fibroblast Basal Medium, supplemented with Fibroblast Growth Kit Serum-Free, Phenol Red and Penicillin-Streptomycin-Amphotericin B Solution (ATCC, Manassas, VA, USA). When appropriate, cells were counted after Hoescht staining (VWR International, Radnor, PA, USA).

HUVEC were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). HUVEC were cultured in vascular cell basal medium (ATCC, USA) supplemented with Endothelial Cell Growth Kit-VEGF and Penicillin-Streptomycin-Amphotericin B Solution (ATCC, USA) (so-called complete culture medium). The cells were cultured at 37 °C and 5% CO₂, with a constant humidity of 95% (Panasonic, Osaka, Japan). Culture medium was freshly changed every 2 days. HUVEC at 4th passage were used for the assessment. HUVEC suspension was prepared by trypsinization and seeded into each well of a 96-well plate at 1 × 10⁴ cells/mL. The culture plate was incubated in 5% CO₂ humid atmosphere at 37 °C overnight for cell attachment and spreading.