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

-tapi-

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.

-bob1-

Hi Bob,

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

-tapi-