Experiments on the isolation of nuclei of Tetrahymena cells
Experiments on the isolation of nuclei of Tetrahymena cells
This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.
Operation method
Isolation of nuclei of Tetrahymena cells
Materials and Instruments
Cells Move 1. Detection of the number of cells used. For more product details, please visit Aladdin Scientific website.
Octanol PMSF
Blender Optical Microscope
2. add protease inhibitors to all solutions while stirring vigorously in a cold environment prior to harvesting cells.
3. add octanol to medium A and mix well.
Add octanol and PMSF to Medium A. This is Medium B.
4. Harvest the cells by centrifugation in a 250 ml conical centrifuge flask with a bucket-type rotary head at 1500-2000 g for 5 minutes at 2°C.
5. Pour out the supernatant quickly and stir to loosen the precipitate.
6. Resuspend the precipitated cells directly in medium B.
Cell Breaking
7. Break the cells by stirring in a cold blender at high speed for 30-60 seconds.
8. Examine the homogenate with a light microscope by placing a small amount of homogenate on a slide and adding methyl green. Ensure that the small nuclei have been removed from their "cups" adjacent to the large nuclei.
Differential nuclear lamellar precipitation
9. Pour the stirred cells into a conical centrifuge flask and centrifuge at 1500-2000 g for 5 minutes at 0-4°C with a bucket-type rotor. Centrifuge bottles of 50-250 ml can be used.
10. Shake the bottle gently to loosen the surface layer formed on the surface of the supernatant and pour the supernatant and surface layer into a cold churn.
11. Resuspend the lamellar precipitate with a small amount of Nucleus Wash Buffer, stain with methyl green and carefully observe the small amount under a light microscope to detect the number of macronuclei and micronuclei.
12. Collect the flake precipitate into a test tube and label it as Pn.
13. Stir the supernatant at high speed for 20 seconds and centrifuge at higher speed for 5 minutes.
14. Collect the supernatant, resuspend the flakes in Nucleus Wash Buffer, and test for nuclei according to step 11.
15. Repeat the agitation and centrifugation, increasing the speed of centrifugation, and analyze the flake precipitate each time until there are almost no large nuclei in the precipitate.
16. When the majority of nuclei are present in the last observed slice, agitate the supernatant at high speed for 20 seconds and centrifuge at 5000 g for 5 minutes at 0-4°C with a bucket rotor.
17. Collect the supernatant, suspend the flake precipitate in Nucleus Wash Buffer, and test the flake precipitate according to step 11.
Count the total number of isolated nuclei
18. Concentrate the appropriate flake precipitates containing large nuclei; do the same for small nuclei. Resuspend the nuclei in the appropriate volume of Nucleus Wash Buffer.
19. Dilute a small amount of large or small nuclei with methyl green. Count the number of nuclei with a spectrophotometer and calculate the number based on the number of starting cells. It is assumed that there is one macronucleus and one mininucleus per cell during growth or starvation; the number of nuclei per cell during splicing varies greatly depending on the stage at which it occurs.
20. Nuclei can be harvested by centrifugation at 2000 g for 5 min at 0-4°C when the amount of macronuclei and/or mininuclei is appropriate.
21. Resuspend nuclei in a small amount of Nucleus Wash Buffer and centrifuge for 5 minutes in a small centrifuge tube to wash the nuclei.