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Cell lysis in chromosome conformation capture

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#1 tapi

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Posted 28 August 2013 - 04:15 AM

Hi all,

 

I am trying to isolate intact nuclei from fixed fibroblasts to perform "chromosome conformation capture" but I have some problems.

 

Protocol includes:

 

- Cells in suspension are fixed (2% PFA).

- Cells are lysed to recover intact nuclei (standard buffer that work for most cells and tissues is hypothonic solution including 0,5% NP-40 and 1,15% Triton)

- Intact nuclei are checked by staining with Methyl-pyronin green and they should appear as round blue nuclei with a little of pink cytoplasmic debris attached.

 

When used with fibroblasts I got only pink staining which is sympthom of inefficient cel lysis. Mechanical forces like douncers also do not help

 

Anyone has suggestions/experience for this problem?

 

Thanks and best regards



#2 bob1

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Posted 29 August 2013 - 05:56 PM

Can you get non-fixed cells?  Fixation cross-links proteins in the cells and will make it hard to separate the nucleus from the cytoplasm.



#3 tapi

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Posted 03 September 2013 - 06:27 AM

Hi Bob,

 

the problem is that fixation is essential for the technique, So I need to overcome this problem







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