Reagents and equipment:
1. OptiPrepTM (600 g/L of iodixanol);
2. Homogenization medium: 0.25 mmol/L sucrose solution, 1 mmol/L ethylenediaminetetraacetic acid, 1 g/L ethanol, 5 mmol/L 3-morpholinylpropanesulfonic acid (MOPS), pH 7.2;
3. Diluent: 6mmol / L ethylenediaminetetraacetic acid, 6g / L ethanol, 30mmol / L MOPS, pH 7.2;
4. 1 mol/L sucrose solution;
5. Add protease inhibitors to the homogenate medium and diluent as required;
6. Prepare the mitochondrial components using a homogenizing medium;
7. Dounce homogenizer (10ml loose preparation, Wheaton B type);
8. Two-chamber gradient preparation instrument or Gradient MasterTM;
9. 5ml syringe (with an inner diameter of approximately 0.8mm) with a metal trocar;
10. Ultracentrifuge with a fixed-angle centrifugal rotor containing a volume of 30-40 ml, a maximum speed of approximately 100,000 g, and a thick-walled tube of approximately 30 ml capacity;
11. Gradient collector;
experimental method:
Mammalian primary cell culture.
After primary cell collection, all operations were performed at 0-4 °C.
1. Prepare a gradient solution using iodixanol, diluent, 1 mol/L sucrose solution and water according to different volume ratios: 500 g/L iodixanol (5+0.6+0.4+0.0), 400 g/L iodine Kesha alcohol (4+0.6+0.7+0.7) and 200g/L iodixanol (2+0.6+1.1+2.3);
2. Resuspend the mitochondrial pellet in a homogenized medium using a loosely prepared Dounce homogenizer (grinding 2-3 times) and adjust accordingly to a volume of 0.5 ml per gram of tissue;
3. Prepare a linear gradient using a two-chamber gradient preparer or Gradient MasterTM, using a syringe and metal trocar from each of the 200 g/L and 400 g/L iodixanol solutions to prepare a gradient solution and place it in a suitable solution. In the polycarbonate tube of the fixed-angle centrifugal rotor, the bottom layer of each gradient is 2 ml of 500 g/L iodixanol;
4. Add 3 ml of the suspension to the upper layer of each gradient solution, and then centrifuge at 105000 g for 1 h;
5. Let the rotor continue to decelerate from 1000r/min. Collect the gradient solution in 1ml. The higher the density, the more you can collect it. Alternatively, a syringe and a metal trocar can be used to collect the peroxisomes forming a band at a distance of 2/3 of the gradient solution;
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