Preparation of nuclei by activator lysis method

Summary

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

Operation method

Preparation of nuclei from suspension-cultured HeLa cells by activator lysis method

Materials and Instruments

HeLa Cells
TM-2 Buffer
Plastic Centrifuge Bottles Corex Tubes

Move

1. Transfer 4 liters of HeLa cells to a concentration of 1. ^0x106 cells/ml.

2. Cells are centrifuged in 1 L plastic centrifuge bottles for 5 minutes at 3000 r/min using a Sorvall RC-3B centrifuge with an H-600A head.

3. In a 30 ml Corex tube, wash each cell sediment with 10 ml of ice pre-cooled PBS.

4. Resuspend the cell sediment with 10 ml of TM-2 buffer and incubate for 1 minute at room temperature.

TM-2 Buffer:

0.01 mol/L Tris-HCl (pH 7.4)

0.002 mol/L MgCl₂

0.0005 mol/L PMSF (added before use)

5. Place the tube containing the TM-2 suspension in a beaker of ice water in an ice bath for 5 minutes.

6. Add Tritron X-100 to a final concentration of 0.5% and ice bath for 5 minutes.

7. Shear the cells 3 times through a 22-gauge needle.

8. Add 5 μl to a 22 ^mm2 glass coverslip and examine the nuclei under a phase contrast microscope.

9. Separate the nuclei from the cytoplasm by centrifugation with a Sorvall RC-5B centrifuge, SS-34 turntable, at 800 r/min for 10 minutes at 4 °C.

10. The nuclei were washed twice with 10 ml of TM-2 buffer.

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