Separation of primary cell lysosomes in discontinuous Nycodenz gradient solution

Separation of primary cell lysosomes in discontinuous Nycodenz gradient solution

Reagents and equipment:
1. Homogenization medium: 0.25mol/L sucrose solution, 1mmol/L ethylenediaminetetraacetic acid, 10mmol/L Hepes-NaOH, pH 7.4;
2. Nycoprep ^TM Universal;
3. Phosphate buffer pH 7.4 (50mmol / L)
4. Nycoprep ^TM Universal diluent: 6mmol / L ethylene diamine tetraacetic acid, 60mmol / L Hepes-NaOH, pH 7.4;
5. Nycodenz working solution (500 g / L): Mix 5 volumes of NycoprepTM Universal and 1 volume of NycoprepTM Universal diluent;
6. Add protease inhibitors to the solution as required;
7. Mitochondrial components;
8. Ultracentrifuge with a bucket rotor with a centrifuge tube of approximately 38 ml capacity (a smaller 17 ml or 13 ml centrifuge tube can also be used);
9.10ml size syringe (about 0.8mm inner diameter) with metal trocar;

experimental method:
All operations were carried out at 0-4 °C.
1. Dilute the Nycodenz working solution with a homogenizing medium to produce a gradient solution of Nycodenz concentrations of 300g / L, 260g / L, 240g / L and 190g / L, respectively, to maintain these solutions at 0-4 ° C;
2. Resuspend the washed mitochondrial pellet in a homogenized medium (4-10 ml) and mix with 2 volumes of Nycodenz working solution;
3. For small tubes of 36-38 ml, use a syringe with a metal trocar in each small tube and add 8 ml 190 g/L Nycodenz to the bottom layer, and take 7 ml each from 240 g/L, 260 g/L and 300 g/L Nycodenz. Add 8-9 ml mitochondrial sediment suspension;
4. Centrifuge at 95000 g for 2 h, then collect lysosomes from 190 g/L and 240 g/L Nycodenz interface or collect all gradient components stepwise before performing labeling analysis;

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