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MB Sample ID: SA214149
| Local Sample ID: | AP02_10 |
| Subject ID: | SU002328 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
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Collection:
| Collection ID: | CO002321 |
| Collection Summary: | To isolate mitochondrial and cytosolic cellular fractions we followed a previously published rapid subcellular fractionation method (Lee et al., 2019). Briefly, on the day of the experiment, cells were washed twice with ice-cold PBS and scraped from the flasks with 2 ml PBS. 1:4 of cells was transferred to a tube and served as the whole cell lysate (WCL). After a brief centrifugation step (10,000 g, 3 minutes, 4 °C), supernatant was removed and 300 ul of metabolite extraction solution (50%MetOH:30%acetonitrile:20% ultrapure water) was added to samples. Samples were incubated with the extraction buffer for 20 minutes in a dry-ice MetOH water-bath and were then subjected to metabolite extraction. The rest 3:4 of cells was subjected to digitonin-based cellular fractionation. More precisely, after a brief centrifugation step (10,000 g, 10 sec, 4 °C), supernatant was removed and the pellet was resuspended in 1 ml of ice-cold digitonin-PBS buffer (1mg/ml). Following quick centrifugation (10,000 g, 10 sec, 4 °C), supernatant and pellet were collected as the cytosolic and mitochondrial fractions, respectively. 4 ml of 50%MetOH:30%acetonitrile was added in the cytosolic fractions to extract metabolites. Samples were incubated for 20 minutes in a dry-ice MetOH water-bath and were then subjected to metabolite extraction. The mitochondrial fraction was resuspended in 100 ul metabolite extraction solution and incubated for 20 minutes in a dry-ice MetOH water-bath and were then subjected to metabolite extraction. |
| Sample Type: | Cultured cells |
