Telotaggg telomerase elisa assay

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).

To measure cytokine release, conditioned media from HEKn cells collected after treatment with melatonin, AFMK or 6(OH)Mel for 24 h were submitted for human cytokine array assay using Proteome Profiler Array kit (R&D Systems). To evaluate L-lactate production in HEKn cell media, the Lactate Assay kit (Colorimetric, Cell Biolabs) was used and the values were recorded using Cytation 5 Imaging Reader. Telomerase activity was measured using the TeloTAGGG Telomerase Elisa assay (Roche) in the cell pellets obtained after 24 h treatment of HaCaT keratinocytes with AFMK, melatonin or 6(OH)Mel at a concentration of 10⁻⁴ or 10⁻⁵ M. The data were collected with Cytation 5 Imaging Reader. All assays followed procedures provided by manufacturers.