need protocol to isolate nuclei from lymphocytes

Kristin B. Andersson kbrevik at u.washington.edu
Thu Oct 20 14:16:08 EST 1994


In my experience, there is a very large variation in sensitivity to
NP40  in various lymphoid cell lines.  In human B cell lines, 0.05%
(yes!!)  NP40 is sufficient to get "functional" nuclei for f.ex.
nuclear runon experiments and isolation of DNA binding proteins.  In my
hands, 0.5% NP40 lysed most of the nuclei in the suspension, resulting
in DNA "goo" in the tube.

My best advice to your question is to titer the NP40 concentration
carefully.

My protocol for generating nuclei in human B cells, T cells and the Reh
cell line goes as follows  (look up Andersson et al, FEBS Letters 1994,
337, 71-76):

20-40 10x6 cells per timepoint (depending on what you are doing)

The procedure is done one ice!
Wash in PBS, pellet at 500xg for 5 min.
Resuspend in 450 ul hypotonic buffer (10 mM Tris, pH7.6, 10 mM NaCl, 3
mM MgCl2)

Omit any swelling step

Add 50 ul 0.5% NP40 (or whatever is the appropriate concentration)
Mix immediately by inversion.

Spin 200xg (NOTE!!  Too fast will crush the nuclei.  All spins after
this step at 200 x g.  NOTE II  This is cell type dependent)  for 5
min.

Wash in 1 ml hypotonic buffer (without NP40).  Pellet nuclei, and use
them for your purpose.

In general, if the pelleted nuclei are difficult to resuspend, it is
usually an indication of lysed nuclei.  Check the nuclei by trypan-blue
exclusion.  The nuclei should not be in clumps (lysis), but rather as a
single "cell" suspension.


Good luck!!!

Kristin

Kristin B. Andersson                    kbrevik at u.washington.edu
University of Washington                Tlf:  +1-206-543-1426
Dept. of Microbiology, SC-42                         685-9992
Seattle, WA 98195                       Fax:  +1-206-685-0305



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