OCR assay
As previously reported, OCR was measured using the MitoXpress® Xtra
probe or Cell Mito Stress Test Kit (Agilent Technologies) by a
time-resolved fluorescence (TR-F) plate reader (Cytation 5; BioTek,
Winooski, VT, USA) or Seahorse XFp analyzer
(Agilent Technologies),
respectively (Yepez et al., 2018). After
incubation with GS or ginsenoside monomer for 48 h, each well was
equilibrated in a CO2-free incubator at 37°C for 1 h and
measured every 2 min over 2 h to obtain TR-F intensity for calculating
the initial slopes (μs min-1), according to the
phosphorescence lifetime (τ) values. To further analyze mitochondrial
respiration, oligomycin (1 µM), FCCP (5 μM), rotenone (Rot, 1 μM), and
antimycin A (AA, 1 μM) were sequentially added to each well with XF base
medium (Agilent, pH 7.4) supplemented with 2 mM L-glutamine, 1 mM
pyruvate, and 25 mM glucose. After normalization to the intensity of
Hoechst 33342 nuclear staining, basal OCR, ATP production, maximal
respiratory capacity (MRC), and spare respiratory capacity (SRC) were
calculated.