Mitochondria isolation kit

RTECs were fractionated into cytosol and mitochondria by using a Mitochondria isolation kit (BioVision, Inc. CA, USA) as per the manufacturer’s protocol. In short, cells were collected and washed with 10 ml ice-cold PBS. Cells were centrifuged at 600 × g for 5 min at 4 °C and resuspended in 1.0 ml of 1× Cytosol Extraction Buffer. Homogenization was performed on ice and centrifuged at 1200 × g for 10 min at 4 °C to remove nuclei and intact cells. The collected supernatant was centrifuged at 10,000 × g for 30 min at 4 °C. The resulting pellets were resuspended in 1.0 ml of 1× Cytosol Extraction Buffer and centrifuged at 10,000 × g for 30 min at 4 °C to obtain mitochondria. The obtained mitochondria were lysed in 30 μl of Mitochondrial Lysis Buffer and added to 100 ul of enzyme mixture (included in the isolation kit) with 100 μl absolute ethanol. After centrifugation, the resulting pellet was mtDNA. The cytosolic mtDNA was obtained from the supernatant after precipitation with ethanol. The concentration of mtDNA was determined by qPCR assay.

To detect cytosolic mtDNA with qPCR, the cytosol of cultured cells was extracted with a Mitochondria Isolation Kit (Biovision Inc., USA) according to the manufacturer’s instructions. Briefly, after washing with ice-cold PBS, cells treated as indicated were then centrifuged at 600 × g for 5 min at 4 °C. The pellet was resuspended in Cytosol Extraction Buffer Mix and incubated for 10 min on ice. The cell suspension was homogenized on ice and centrifuged at 1200 × g for 10 min at 4 °C to remove nuclei and unbroken cells. The supernatant was transferred to a fresh 1.5 ml tube and centrifuged at 10,000 × g for 30 min at 4 °C. The supernatant was collected as cytosolic fraction. The mtDNA was detected by qPCR with primers that hybridized to sequences in the gene encoding mitochondrial cytochrome c oxidase 1 (mt-Co1). The nuclear DNA was measured with qPCR using primers that hybridized sequences in the 18 S rDNA (coding 18 S ribosomal RNA). The primers for human mt-Co1 were 5′- TTCTTATTTACAGTTGGTGGTC-3′ (forward) and 5′-TCTGAGTAGCGTCGTGGT-3′ (reverse). The primers for 18 S rDNA were 5′-GTAACCCGTTGAACCCCATT-3’ (forward) and 5′-CCATCCAATCGGTAGTAGCG-3′ (reverse). The copy numbers of mtDNA were normalized against the copy numbers of nuclear DNA, and compared between the groups.

Cells were seeded and dissolved by 100 μl ice cold PDH Assay Buffer. Mitochondria from the cultured cells were isolated using a Biovision’s Mitochondria Isolation Kit. According to the protocols of Pyruvate Dehydrogenase Activity Colorimetric Assay Kit(Biovison, San Francisco, USA), 0, 2, 4, 6, 8 and 10 μl of 1.25 mM NADH Standard were added into the wells of 96-well plate and Color densities were measured (OD 450 nm) using a microplate reader (Tecan, Infinite M200, Switzerland) for 10–60 min at 37 °C. Then plot of the NADH Standard Curve used the protein concentration as standardization.