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MB Sample ID: SA337373
| Local Sample ID: | S09_pulldown_Apoptosis_31.09 |
| Subject ID: | SU003230 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
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Collection:
| Collection ID: | CO003223 |
| Collection Summary: | Human osteosarcoma U2OS WT, U2OS BAK Ko expressing GFP BAK, and U2OS FADS2 KO cell lines were cultured at 37 °C and 5% CO2 in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (Invitrogen, Germany). For lipidomic experiments cells were incubated with 1 μM of ABT-737 and S63845 in the complete media and incubated for 50 min at 37°C and 5% CO2. FADS2 KO in U2OS cells was generated in the lab by the CRISPR/Cas9 method. Linoleic acid stock (50 mM) was prepared in ethanol and diluted into culture media before adding them to the cells. Mitochondria were isolated from cultured human osteosarcoma cells by mechanical disruption of cells followed by differential centrifugation: Cells were harvested by trypsinization, washed in PBS, and then resuspend in isolation buffer (IM;250 mM sucrose, 5 mM Tris, and 2 mM EDTA; pH 7.4 and protease inhibitor cocktail) and mechanically broken using glass homogenizer on ice (30-40 strokes on ice) and total cellular lysates were spin down first to remove nuclei and cell debris at 600 x g for 5 min and later at 10,800 x g for 10 min at 4°C to get the crude mitochondria. Mitochondrial pellet was washed 2-3 times with isolation buffer to remove other impurities from mitochondria. Isolated mitochondria were solubilized using SMA co-polymer. For this, mitochondria either from apoptotic or healthy cells were incubated with 0.5% SMA (2:1) for 45 min at room temperature with gentle rotation. Mitochondrial membrane was spun down at 100,000 x g for 40 min to separate solubilized SMALP from the insolubilized membrane. Next, the size of SMALP was analyzed by Dynamic Light Scattering (DLS). For DLS measurements, 15 μl of sample was added to a quartz cuvette which had been thoroughly cleaned with Milli-Q H2O. The cuvette was placed in DynaPro NanoStar (Wyatt Technology corporation, USA) and the sample was analyzed using 10 runs with 10 second acquisition time. This helps to determine the mass distribution of the sample as well as the estimated size of the particles. The distance distribution is shown on a log scale. The size of SMALP as well as the homogeneity with in the sample were also checked by Negative Transmission Electron Microscopy (TEM). For this the diluted SMALPs were placed onto a glow-discharged copper grid (Electron Microscopy Sciences) coated with a layer of thin carbon, washed twice with water, stained with 2% uranyl acetate for 5 min and then air-dried. The grids were imaged on a JEOL JEM2100PLUS electron microscope and recorded with a GATAN OneView camera (CECAD Imaging Facility). mEGFP-BAK-SMALPs were affinity purified from total solubilized mitochondrial membrane fraction (SMALP). For this total SMALP were incubated with 25 μl of GFP-trap MA beads for 90 min with slow rotation in cold room. Beads were washed 2 times with 100 μl of Tris buffer (50 mM Tris 150 mM NaCl pH 8), and finally resuspend in 100 ul of Tris buffer. Small aliquots of unbound and wash fractions were used to analyze the purification quality. |
| Sample Type: | Mitochondria |
