Preparation of cell nuclei by osmotic lysis

Summary

This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.

Operation method

Preparation of nuclei from HeLa cell suspension cultures by osmolysis

Materials and Instruments

HeLa Cells
Earle's Balanced Salt Solution RSB
Glass Coverslip

Move

1. Suspend HeLa cells in culture and centrifuge at 600 g for 5 minutes to collect the cells.

2. Sediment cells are resuspended in 5x volume of pre-cooled Earle's Balanced Salt Solution at 4°C.

3. Resuspended cells are centrifuged at 4°C, 600 g for 5 minutes or longer as described in step 1.

4. Resuspend washed cell sediment with 10x volume of RSB and allow tube to stand on ice for 10 minutes.

RSB:

0.1 mol/L NaCl

1.5 mmol/L MgCl₂

0.01 mol/L Tris-HCl (pH 7.4)

5. Stick a Pasteur pipette tip 0.5-0.75 cm into the cell suspension and apply a small amount of cells onto a slide by siphoning.

6. Cover with a 22 ^mm2 glass coverslip and examine the cells under a phase contrast microscope.

7. Siphon the swollen cells into a pre-cooled glass Dounce homogenizer.

8. Extract the pestle and mortar upward 10 to 12 times rapidly to break up the cells.

9. Take a small amount of the cell suspension and observe it under a phase contrast microscope (same procedure as step 6).

10. After the ratio of free nuclei to intact cells in the homogenized product of the cell suspension reaches or exceeds 9.0, centrifuge the suspension at 1000 g for 3 minutes at 4°C to precipitate the nuclei.

11. Remove the supernatant with a Pasteur pipette fitted with a rubber bulb, and resuspend the nuclei in a 10-fold volume of RSB with a clean Pasteur pipette.

12. Centrifuge the nuclei as in step 10 and resuspend in 10x volume of RSR.

13. Repeat step 12.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/