Oxygen consumption rate
Oxygen consumption rate was performed as before (Wei et al., 2020). In
short, 5×106 immortalized lymphocytes from the
candidate and the age-matched healthy controls were rapidly and gently
harvested and then added to the oxygraphy-2k detector (Oroboros,
Austria). Oligomycin (0.1 mg/mL, Sigma-Aldrich) was used to detect the
ATP-linked respiration. Carbonyl cyanide 4-(trifluoromethoxy)
phenylhydrazone (0.1mM, Sigma-Aldrich) was for the maximal respiration
and the reserve respiratory capacity.
Cellular ATP content and
mitochondrial ROS level detection
Cellular ATP and mitochondrial ROS content were measured by ATP
bioluminescent assay kit (Sigma-Aldrich) and MitoSOX™ Red reagent
(Thermo Fisher Scientific) following the manufacturer’s instruction,
respectively (Wei et al., 2020). For cellular ATP content measurement,
immortalized lymphocytes from patient and age-matched healthy controls
were collected and s resuspended with filtered ultrapure water. Then
added the cell suspension to the ATP-releasing solution and detected the
autofluorescence. The protein concentration of the samples was measured
with the BCA protein concentration assay kit (Thermo Fisher Scientific)
as a calibration. For the mitochondrial ROS level detection,
immortalized lymphocytes from patients and age-matched healthy controls
were collected and resuspended in working solution containing 5uM
MitoSOX reagent. Then incubated at 37°C for 10 mins at dark, gently
washed cells and detected the fluorescence.