Agilent seahorse xf96 extracellular flux analyzer

The mitochondria energy metabolism of TM3 cells was determined with Agilent Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Agilent Technologies). TM3 cells were seeded at a density of 6 × 10³ cells/well in XF96 Cell Culture Microplates. After 24 hours with PA (0, 0.2 and 0.4 mmol/L) treatment, cells were washed with XF base medium supplemented with 2.5 mmol/L glutamine, 0.05 mmol/L sodium pyruvate and 17.5 mmol/L glucose, in which pH was adjusted to 7.4 at 37°C, and then, cells were incubated in a CO₂‐free incubator at 37°C for 1 hour. Seahorse Cell Mito Stress Test was performed to measure oxygen consumption rate (OCR) with a sequential addition of inhibitors of mitochondrial function: oligomycin (2 μmol/L), carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP, 1 mmol/L) and a combination of rotenone and antimycin A (0.5 mmol/L). The mitochondrial OCR in basal respiration, ATP production, maximal and spare respiration was monitored with analyser. Results were normalized after adjustment for the corresponding total protein content per well.

Primary human keratinocytes were isolated from skin biopsies of healthy controls as previously described [44 (link)]. Keratinocytes were seeded at 0.25 × 10⁵ cells/well overnight on Seahorse 96-well XFe96 microplates in culture medium. The next day, cells were treated with melatonin, AFMK and 6(OH)Mel for 24 h, all at a final concentration of 10⁻⁵ M. Just before the start of the experiment, cells were depleted of glucose for 1 h in a 37 °C non-CO₂ incubator. Oxygen consumption rate (OCR or mitochondrial respiration) and extracellular acidification rate (ECAR or glycolytic function) in live cells in real-time were measured using the XF Cell Mito Stress Test and Agilent Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA). The Cell Mito Stress Test media was supplemented with 2 mM GlutaMAX™ Supplement, 1 mM sodium pyruvate, and 25 mM glucose and injected according to the Mito Stress Test. Results are automatically generated from Wave data that was exported to Excel.