Reagents and equipment:
1. homogenization medium: 0.25mol/L sucrose solution, 1mmol/L ethylenediaminetetraacetic acid, 10mmol/L Hepes-NaOH, pH 7.4;
2. Add protease inhibitors to the solution as required;
3. Phosphate buffer;
4. Low-speed refrigerated centrifuge with a bucket-type centrifugal rotor with a 30-40 ml centrifuge tube;
5. High-speed centrifuge with fixed angle rotor with 30-40ml centrifuge tube;
6. For liver: Potter-Elvehjem (polytetrafluoroethylene resin and glass) homogenizer (30-40ml), connected to high torque bridge motor (semiconductor switch control);
7. For cells: ball bearing homogenizer or syringe with a 23 or 25 gauge needle;
8. Dounce homogenizer (loosely formulated, Wheaton B type);
9. 20ml size syringe (about 0.8mm inner diameter) with metal trocar;
experimental method:
All operations must be carried out at 0-4 °C.
1. For the liver: Use very finely shredded tissue with scissors (or use a tissue chopper) and transfer to a Potter-Elvehjem homogenizer with homogenized media (10 ml medium per 2.5 g of tissue). Grinding the mortar about 6 times to homogenize (500-700r/min);
2. For cells: Wash (1 - 3) × 108 cells in 5 ml of phosphate buffer and wash with homogenized medium. The cells were suspended in 3 ml homogenized medium and homogenized 5 times in a ball bearing homogenizer;
3. Centrifuge at 800g for 10min to precipitate nuclear, cell debris and unbroken cells;
4. Pour or aspirate (using a syringe and syringe) the supernatant and place on ice;
5. Grind 2-3 times in a loosely prepared Dounce homogenizer for homogenization, and resuspend the pellet using 10 ml homogenate medium (5 ml for cells);
6. Repeat centrifugation and combine the supernatants;
7. Centrifuge the combined supernatant at 3000g for 10min;
8. Centrifuge the supernatant obtained in step 7 at 15000 g for 10 min;
9. Resuspend the light mitochondrial pellet with 10 ml (cell corresponding to 5 ml) homogenate medium, and gently grind it 2-3 times with a mortar in a loosely prepared Dounce homogenizer;
10. Centrifuge at 15000g for 10min;
11. Resuspend the pellet in an appropriate medium for analysis or further processing;
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