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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