Mitochondria Isolation Kit for Cultured Cells

1. Isolation of mitochondria from cultured cells

1. Collect approximately 2-5 × 10⁷ cultured cells and wash twice with pre-cooled PBS.

2. Resuspend cells in 1 mL pre-cooled Mitochondria Isolation Reagent supplemented with PMSF to a final concentration of 1 mM, and incubate on ice for approximately 10-15 min.

3. Homogenize the cells on ice with a glass homogenizer. Trypan Blue may be used to monitor membrane disruption; adjust the number of strokes according to cell type.

4. Centrifuge at 600 × g for 10 min at 4°C to remove nuclei, unbroken cells, and large debris.

5. Carefully transfer the supernatant to a new tube and centrifuge at 11,000 × g for 10 min at 4°C.

6. Carefully remove the supernatant. The pellet contains mitochondria.

Note: To obtain mitochondria-free cytoplasmic protein, further centrifuge the supernatant from Step 5 at 12,000 × g for 10 min at 4°C and collect the final supernatant.

2. Downstream use of isolated mitochondria

For intact mitochondria, gently resuspend the pellet in an appropriate amount of Mitochondria Storage Buffer. For mitochondrial protein extraction, lyse the pellet with Mitochondria Lysis Buffer containing 1 mM PMSF for BCA assay, SDS-PAGE, Western Blot, IP, or enzyme activity assays.

Note: Perform all isolation steps at 4°C or on ice and use pre-cooled reagents.