Xf24 well microplate

Isolated NK cells were cultured for 7–10 days in the presence of 1,000 units/ml recombinant human IL-2 and treated with or without 30 ng/ml IL-18 for 24 hours. XF 24-well microplates (Seahorse Bioscience) were precoated with Cell-Tak (Corning) for 2 hours before seeding the NK cells on the plate for real-time analysis of the Oxygen consumption rate (OCR). 10⁶ NK cells were cultured per well, and various inhibitors were added (Agilent Seahorse XF Cell Mito Stress Test) at the following concentrations: oligomycin (2 μm), FCCP (0.5 μm), and rotenone (100 nM) plus antimycin A (4 μM), which allow the accurate calculation of respiration capacity (OCR).

C2C12 myoblasts were seeded in XF 24-well microplates (Seahorse Bioscience, Billerica, USA) and differentiated. Mitochondrial inhibitors, including 1 μmol/L oligomycin, 1 μmol/L carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 1 μmol/L rotenone/antimycin A, were incubated with these cells respectively. The cell OCR was measured by the extracellular flux analysis (Seahorse Biosciences) every 8 min.
Basal respiration represents the baseline oxygen consumption reading prior to the addition of inhibitors, antimycin A Rotenone, with the subtraction of non-mitochondrial respiration. Maximal respiration is shown as the maximum OCR measurement value following FCCP injection and with the subtraction of non-mitochondrial respiration. ATP production respiration represents the OCR reduction after the addition of an inhibitor of ATP synthase, oligomycin. Spare respiratory capacity is calculated by maximal respiration subtracting basal respiration. After detection, cell protein content was calculated, and OCR was adjusted accordingly.

mIPA cells were plated in Seahorse XF 24-well microplates and fully differentiated for 6 days. After incubation with 10 µg/mL of EEs for 6 h, mitochondrial stress test was performed by treating cells with 2 µM of oligomycin, 1 µM of FCCP, and 1 µM of rotenone/antimycin A, sequentially followed by measurement of energy metabolism of live cells using an XF24 Analyzer (Agilent, Santa Clara, CA, USA), according to the manufacturer’s instructions.

To further analyze mitochondrial metabolic functions in live PD-MSCs^PRL-1, an XF24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA) was used for real-time analysis of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Naïve and PD-MSCs^PRL-1 were plated on XF24-well microplates (Seahorse Bioscience) (7 × 10³ cells/well). The cells were adjusted for equilibrium in XF buffer for 60 min for contemporaneous analysis of OCR and ECAR by repeated cycles of mixing (3 min), incubation (2 min), and measurement (3 min) periods in a non-CO₂ incubator. Following basal respiration measurements, the cells were sequentially treated with 0.5 μM oligomycin, 0.5 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and 1 μM rotenone/antimycin A (AA) mixture. The changes in respiration were recorded. The Seahorse XF24 Analyzer program was set according to the manufacturer’s recommendation. OCR/ECAR was then normalized by total cell number.