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Cell lysis in chromosome conformation capture - (Aug/28/2013 )

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


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.


Hi Bob,


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