How to Lyse Primary Cells from Tissue
Biochemistry, Molecular Biology, and Cell Biology Protocols >> How to Lyse Primary Cells from Tissues
Solutions required :
- 10mM Tris, 2mM EDTA (TE buffer)
- 2% Triton in PBS
1. Wash the tissue in a relatively large volume of PBS (eg 5-6 times the volume of the tissue) 3-4 times by centrifugation at 4°C, 10 minutes each, at 10,000 x g. Make sure that the tubes are balanced when centrifuging.
2. After the first centrifugation, most of the cells can probably be separated from the "guey" material of the extracellular matrix by selectively pipeting the cells up and down in PBS, leaving the rest of the tissue at the bottom of the centrifuge tube, and transferring the cells to a new centrifuge tube.
2. If necessary, dissociate the cells from each other by adding a small amount of collagenase or trypsin for a small amount of time on ice. Stop the dissociation reaction with inhibitors (eg TIMP for collagenase, or fetal calf serum for trypsin).
3. Centrifuge the cells down one more time for 10 minutes at 10,000 x g, remove the supernatant, and then resuspend cells in the pellet with TE buffer
2. Wash 2x in TE (spin at 40,000 x g, 8 minutes each time). Make sure that the tubes are balanced when centrifuging.
3. To 2% Triton X-100 in PBS while stirring, add the cells dropwise resuspended in ~1mL TE (try to keep the solution on ice). Other detergents can also be used, such as 18 mM CHAPS.
4. Solubilize for 30 minutes in the cold room after added cells dropwise
5. Spin at 100,000 x g, 10 minutes or longer. Make sure that the tubes are balanced when centrifuging.
6. Take the supernatant of the lysed cells containing the cellular proteins.
7. Alternatively, for tissues containing a lot of lipids, instead of solubilizing the cells with detergent, it may be necessary to extract out the lipids. However, by extracting the lipids in this condition, the proteins may be denatured. To extract the lipids, add chloroform:methanol in a 1:2 ratio to the tissue, vortex, then add a 1:1 ratio of chloroform:water, vortex again, and then and centrifuge at 1000 x g for 10 min. The top aqueous phase contains the proteins from the lysed cells (the proteins may be denatured), and the bottom organic phase contains the lipids.