Dear All
We are trying to isolate mitochondria from human skin fibroblasts by applying various protocols involving douncing. We tried to use nearly 50 million cells, used different douncers, and dounced at different rates as well. Yet, it seems the cells do not break completely, and we end up with 50 ug impure mitochondria at best.
Any suggestions are welcome. Alternatively, is there a protocol that employs a method other than douncing to break the cells?
Regards
Sahil
Isolating mitochondria from human skin fibroblasts involves several steps to break open the cells (homogenization), separate the mitochondria from other cellular components, and purify the mitochondria. Here's a basic protocol for isolating mitochondria from human skin fibroblasts:
Materials:
- Human skin fibroblasts
- Mitochondria isolation buffer (e.g., sucrose-based buffer)
- Homogenization buffer (e.g., Tris-based buffer)
- Differential centrifugation tubes (e.g., conical tubes)
- Centrifuge capable of high speeds (e.g., ultracentrifuge)
- Ice and ice bucket
- Pipettes and tips
- Microcentrifuge tubes
- Protein assay kit for quantification
Protocol:
By following this protocol, you can isolate mitochondria from human skin fibroblasts for further analysis of mitochondrial function, protein composition, or other mitochondrial studies. Adjustments to the protocol may be necessary based on the specific requirements of your experiments and the characteristics of the fibroblast cell line used.
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