Nusse Nuclear Preparation

This protocol will isolate nuclei stained with EthidiumBromide (you can subsitute PropidiumIodide if you prefer) for cell cycle analysis by flow cytometry.

1. harvest cells (with trypsin/EDTA for adherent cells) and spin down.
   
   • if you are in a tremendous hurry, you can aspirate the media from adherent cells and add Solution I directly to the plate
     
2. rinse cells in PBS and respin, aspirate supernatant
   
   • optional
     
3. resuspend gently in Solution I (use 1 ml for 1-2 million cells)
   
   • immediately before use add 2.5 μl ethidium bromide (10 mg/ml) and 1 μl RNaseA (10 mg/ml) per ml of Solution I
     
4. let sit at room temperature for about an hour
   
   • this gives the RNAse time to work. the incubation can be much shorter (2-3 minutes) if you are in a hurry and not worried about RNA messing up your cell cycle profile, or if you intend not to analyze the nuclei right away
     
5. gently mix in an equal volume of Solution II
   
   • immediately before use add 4 μl ethidium bromide (10 mg/ml) per ml of Solution II
     

The nuclei can be stored at 4C for 2-3 days.

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Solutions

Solution I	final []
292 mg NaCl	10 mM
500 mg Sodium Citrate ⋅ 2H2O	3.4 mM
150 μl Nonidet P-40 NOT Tergitol type NP40	0.03%
add H2O to 500 ml	store at 4C

Solution II	final []
7.5 g Citric Acid (8.2 g of citric acid monohydrate)	78 mM
42.7 g Sucrose	250 mM
add H2O to 500 ml	store at 4C

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References

Giaretti & Nusse, Methods in Cell Biology 41, p389-400 (1994).
Nusse et. al, Cytometry 11, p813-821 (1990).

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Comments

• Solution I is hypotonic to swell cells while the light detergent gently breaks the plasma membrane. Nuclei are recoverable at this stage, but carry a lot of debris
  
• Solution II is isotonic. The citric acid delaminates the nuclear membrane releasing the debris from clean nuclei. The pH of solution II is very low, ~2 or so.