I need to extract cell nuclei from animal tissue before DAPI staining for flow cytometry analysis. I need to test a lot of samples, so I'm looking for a cheaper alternative to ready-made kits. Does anyone know a recipe for such a buffer?
Hello Katarzyna Grecka
You may try the recipe given below.
Nuclei Isolation Buffer Recipe:
15mM Tris-HCL (pH 7.5)
60mM KCl
15mM NaCl
5mM MgCl2
1mM CaCl2
250mM Sucrose
You may add protease and phosphatase inhibitor cocktail to the buffer fresh each time you use the buffer for nuclei isolation.
Brief protocol:
You may cut the tissue into small cubes, and then gently use a dounce homogenizer to more fully dissolve the tissue cubes in the nuclei isolation buffer. Add nuclei isolation buffer containing 0.3% NP-40 to the cut tissue cubes. This should be a cloudy homogenous tissue milkshake when you have finished. Pellet the nuclei at 600 rcf for 5 min at 4°C (nuclei pellet should be smaller and much whiter in color than the whole cells). Resuspend the nuclei pellet gently with nuclei isolation buffer but without NP-40 detergent for washing. Finally, pellet nuclei at 600 rcf for 5 min at 4°C and discard the supernatant.
This should be a much cheaper alternative.
Hope it may be helpful!
Best.
- Badges
- Science topic
- Similar topics
- Medicine
- Epidemiology