After primary cell culture, the main organelles were analyzed by iodixanol gradient solution.
Reagents and equipment: experimental method: Chef Buddy Kitchen,High Quality Chef Buddy Kitchen,Chef Buddy Kitchen Details, CN Eastony Industries (NingBo) Co., LTD. , https://www.eastony.com
1. OptiPrepTM (600 g/L of iodixanol);
2. Homogenization medium: 0.25 mmol/L sucrose solution, 1 mmol/L ethylenediaminetetraacetic acid, 20 mmol/L Hepes-NaOH, pH 7.4;
3. OptiPrepTM dilution: 0.25mmol/L sucrose, 6mmol/L ethylenediaminetetraacetic acid, 120mmol/L Hepes-NaOH, pH 7.4;
4. Iodixanol (500g/L) working fluid (IWS): 5 volumes of OptiPrepTM +1 volume OptiPrepTM dilution;
5. Add protease inhibitors to the homogenate medium and the OptiPrepTM dilution as required;
6. A light mitochondrial component prepared using a homogenizing medium;
7. Dounce homogenizer (loosely formulated, Wheaton B type);
8. Two-chamber gradient preparation instrument or Gradient MasterTM;
9. 5ml size syringe (with an inner diameter of approximately 0.8mm) with a metal trocar;
10. Ultracentrifuge with bucket rotor (centrifuge tube capacity 17ml);
11. Gradient collector for collecting low density or high density gradient liquid first;
After primary cell culture, all operations were performed at 0-4 °C.
1. Dilute the iodixanol working solution with a homogenizing medium to obtain a gradient solution of iodixanol concentration of 190 g/L and 270 g/L, and place the solution on ice;
2. Resuspend the light mitochondrial pellet in a 3.0 ml homogenized medium using a loosely prepared Dounce homogenizer (milled 2-3 times with a mortar);
3. Adjust the concentration of iodixanol in the suspension to 300 g/L using iodixanol working solution;
4. Prepare a linear gradient using a two-chamber gradient preparer or Gradient MasterTM from each of the two gradient solutions, placed in a tube that can be centrifuged in a bucket rotor;
5. Using a syringe and metal trocar, place 3-4 ml of the suspension prepared in step 3 under gradient solution;
6. Spread 1-2 ml homogenization medium on the top of the gradient solution so that the liquid level in the tube is about 3 mm from the top, and centrifuge at about 70,000 g for 1.5-2 h in a suitable bucket rotor;
7. The low-density fraction of the gradient solution can be collected by first replacing it with a high-density medium or by sucking it out on the concave surface. Alternatively, the high density component is collected first by pipe piercing or carefully using a thin metal trocar to probe into the bottom of the tube (connected to the peristaltic pump);