Epigro human epidermal keratinocyte complete culture media kit

Neonatal Human Epidermal Keratinocytes (HEKn, primary cells) were isolated from foreskins collected at the Woman and Infant Hospital (UAB) and cultured on Petri dishes (TPP) in EpiGRO™ Human Epidermal Keratinocyte Complete Culture Media Kit (Millipore Merck KGaA, Darmstadt, Germany) as described previously [12 (link)]. Cells were seeded into 24-well plates, maintained until they reached 70% confluency, and stimulated for 24 h with melatonin, 6(OH)Mel or AFMK at the final concentration of 10⁻⁵ M or with solvent only (control, 0.1% EtOH). HaCaT cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) media containing 5% charcoal stripped FBS followed by treatment with 10⁻⁴ or 10⁻⁵ M melatonin and 10⁻⁵ M 6(OH)Mel and 2-hydroxymelatonin (2(OH)Mel) at 37 °C under 5% CO₂ for 6 and 24 h. We have used similar doses of ligands in our previous studies, which demonstrated the paracrine mechanism of action secondary to production and metabolism of melatonin in the epidermis [12 (link),13 (link),29 (link),30 (link)]. After 6 and 24 h, cells were collected in lysis reagent and RNA isolated using Absolutely RNA Miniprep Kit (Agilent Technologies, Santa Clara, CA, USA).

2-Hydroxymelatonin (2(OH)Mel), 6-hydroxymelatonin (6(OH)Mel), bovine serum albumin (BSA), ethanol (EtOH), glucose, melatonin, and sodium pyruvate were purchased from Sigma (St. Louis, MO, USA). AFMK and GlutaMAX™ Supplements were purchased from Cayman Chemical (Ann Arbor, MI, USA) and Thermo Fisher Scientific (Waltham, MA, USA), respectively. Other reagents were supplied as follows: EpiGRO™ Human Epidermal Keratinocyte Complete Culture Media Kit (Millipore Merck KGaA, Darmstadt, Germany); RNA Miniprep Kit (Agilent Technologies, Santa Clara, CA, USA), high capacity cDNA Reverse Transcription Kit (Applied Biosystems), DyNamo Flash SYBR Green qPCR Kit (Thermo Scientific, Waltham, MA, USA), KAPA SYBR^® FAST qPCR Kit (Kapa Biosystems, Wilmington, MA, USA), the Cell Mito Stress Test media was supplemented with 2 mM GlutaMAX™ Supplement (L-alanyl-L-glutamine dipeptide in NaCl), Proteome Profiler Array kit (R&D Systems, Minneapolis, MN, USA), lactate colorimetric assay kit (Cell Biolabs, Inc. San Diego, CA, USA), TeloTAGGG Telomerase Elisa assay (Roche, Basel, Switzerland), and a set of human primers for real-time PCR (Eurofins Genomics, Ebersberg, Germany).

Human primary corneal epithelial cells were axenically cultured in an EpiGRO™ Human Epidermal Keratinocyte Complete Culture Media Kit (Merck Millipore, Darmstadt, Germany). The human primary corneal epithelial cells were cultured to 3 × 10⁵ cells/mL in 24-well plate cell culture dishes. After 24 h of growth, Acanthamoeba live cells at a concentration of 3 × 10⁵ were co-cultured with human primary corneal epithelial cells. Acanthamoeba spp., at a density of 1.0 × 10⁶ parasites/mL in PBS for 4 h, as well as their secreted proteins, were collected. The Acanthamoeba-secreted proteins (150 μg) were co-incubated with human primary corneal epithelial cells for 2 h, followed by microscopy observations and CPE assays. The cells were fixed in wells with methanol with acetic acid at 3:1 for 15 min. After air drying, we added 1 mL Giemsa Buffer:Giemsas Azur-Eosin-Methylenblaulosung stain buffer (9:1) in a fixed well for 15 min. The wells were rinsed three times with ddH₂O and then left to air dry.