Mitochondria isolation kit for cultured cell

Mitochondria Isolation Kit for cultured cells (#89874, Thermo Scientific) was used to isolate cytosolic/ER and mitochondrial fractions. Briefly, 8 × 10⁶ pelleted cells were resuspended with Mitochondria Isolation Reagent A, vortexed for 5 s, and then incubated on ice for 2 min. After adding Mitochondria Isolation Reagent B, samples were vortexed for 5 s, and incubated on ice for 5 min. Mitochondria Isolation Reagent C was then added and the tubes were inverted several times to mix. The supernatant obtained by centrifugation at 3200 rpm for 10 min at 4 °C was transferred to a fresh tube and centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatant containing cytosolic/ER fraction was transferred to a new tube. Mitochondria Isolation Reagent C was added to the pellet, centrifuged at 13,000 rpm for 5 min, and the supernatant was discarded. The pellet containing the mitochondrial fraction was resuspended in 1× SDS loading buffer and analyzed by immunoblot analysis.

Isolation of extracellular mtDNA from A549 and human lung tissue stimulated with the indicated amounts of PLY was done after 3 and 8 hours, respectively. The supernatant containing the mtDNA was collected and centrifuged at 2100 × g for 10 min to remove cell debris. For microvesicle isolation, A549 were treated with PLY (50 or 100 ng/ml) in serum-free media for 4 hours. The microvesicles were isolated from the conditioned media as previously described with minor modifications^{60 (link)}. Microvesicles were washed with PBS and processed for mtDNA analysis. Mitochondria were isolated using the Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher, Darmstadt, Germany) as described by the manufacturer. mtDNA was isolated using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Quantitative PCR was performed using TaqMan assays (MT-CYB, Hs 02596867_s1, Life Technologies) on an ABI 7300 instrument. The amount of mtDNA in the supernatant was calculated based on a standard curve generated using purified mtDNA from isolated mitochondria. mtDNA copy number analysis was performed usingTaqMan assay for mtDNA and nuclear DNA (GAPDH Hs 99999905_m1, Life Technologies). Relative mtDNA copy number was calculated as the ratio of mtDNA abundance versus nuclear DNA abundance and normalized to the control group.

After the respective incubation periods as per the experiments, cells were harvested with the help of 1x PBS. From there on mitochondrial protein extraction was performed as per the manufacturer’s instruction. (Mitochondria Isolation Kit for Cultured Cells, Cat. 89874, ThermoFisher).

The mitochondrial fraction of the harvested brain tissues and cells was obtained according to the previous method 32 (link). The cytosolic and mitochondrial parts of the ischemic penumbra and cells were processed with a Mitochondria Isolation Kit for Cultured Cells in accordance with the manual (provided by ThermoFisher Scientific, America). The lack of cytoplasmic calnexin in Western blotting analysis (data concealed) showed efficient isolation of mitochondria.