Dermalife k keratinocyte medium complete kit

Experimental procedures and methods were approved and performed in accordance to the guidelines of the Institute of Medical Biology, Institutional Biosafety Committee (IMB IBC). Primary human dermal keratinocytes were obtained with informed consent from 4 different donors. Tissue collection and cell banking at the Institute of Medical Biology was approved by SingHealth Institutional Review Board. Experiments were approved by the National University of Singapore Institutional Review Board. Keratinocytes were maintained under standard conditions (37 °C, 5% CO₂) in DermaLife K Keratinocyte Medium Complete Kit (Lifeline Cell Technology) supplemented with 50 U/ml penicillin and streptomycin (Invitrogen). For UV irradiation experiments, primary keratinocytes were seeded on 6 cm dishes, incubated for 24 hours and washed once in phosphate buffered saline pH 7.4 before exposed to UV irradiation using a UV lamp (Ultra-Vitalux 300, Osram). UVB flux was measured by a UVB digital radiometer (Model HD2302.0, Delta Ohm). Either non-SPF base cream or SPF50+ sunscreen cream (Neutrogena Ultra Sheer Pure-Mild) was applied on UV-transparent plastic lids (HelioScreen, HD6, Labsphere), left to absorb for 15 minutes and placed over the 6 cm dish during UV irradiation. After exposure, PBS was replaced by keratinocyte medium and cells were incubated for another 3 days.

Peptide DJK-5 (VQWRAIRVRVIR-NH₂; all D amino acids as indicated in italics) was synthesized by CPC Scientific (Sunnyvale, CA, USA) using solid-phase 9-fluorenylmethoxy carbonyl chemistry and purified to ~95% using reverse-phase high-performance liquid chromatography. Peptide identity was confirmed by mass spectrometry. N/TERT cell and skin culture medium including Keratinocyte-SFM medium, Dulbecco’s Modified Eagle Medium (DMEM, high glucose, GlutaMAX™ Supplement, pyruvate), and Ham’s F-12 Nutrient Mix were purchased from ThermoFisher Scientific (Waltham, MA, USA). CnT-Prime 3D Barrier Medium was obtained from CELLnTEC Advanced Cell Systems AG (Zurich, Switzerland). DermaLife K Keratinocyte Medium Complete Kit was purchased from Lifeline Cell Technology (Oceanside, CA, USA). Supplements for skin culture medium including isoproterenol, hydrocortisone, bovine insulin, selenious acid, l-serine, l-carnitine, bovine serum albumin, palmitic acid, linoleic acid, and arachidonic acid were obtained from Sigma-Aldrich (St. Louis, MI, USA). Antibiotic controls (gentamicin and fusidic acid) and 10% neutral-buffered formalin solution were also purchased from Sigma-Aldrich. Reagents for bacterial culture and resuspension including tryptic soy broth (TSB), Luria broth, d-glucose, and phosphate-buffered saline (PBS) were purchased from ThermoFisher Scientific.