Nuclear extracts/suspensions

Kevin Mulcahy K.Mulcahy at sheffield.ac.uk
Tue Nov 5 12:05:19 EST 1996


I've never heard of this method before, but it sounds like it may not be 
working. Have you tried looking at the "nuclei" under the light 
microscope to see if there is anything there resembling nuclei? If so, 
then you could also consider making some electron microscope sections to 
confirm this (and possibly doing indirect immunofluorescence staining 
too).

Aside from this method, in the past I have routinely used a method 
described by Bunce et al (1988) entitled "A rapid procedure for 
isolating hemopoietic cell nuclei" (Analytical Biochemistry, Vol 175: 
67-73). I know this method was developed for haemopoietic cells, but it 
might be worth trying it out on CHO cells (you may have to alter some of 
the conditions though). 

Essentially, the method involves washing the cells (50 million) three 
times in pre-chilled nuclear preparation buffer (NPB; 10mM Tris-HCl, pH 
7.4, 2mM MgCl2, 140mM NaCl), resuspending them in 1ml of NPB containing 
2% (v/v) Tween 40, quickly transfering them to a 1.8ml polycarbonate 
microcentrifuge tube and freezing the suspension by immersing them in 
liquid nitrogen for approx 30 seconds. The cells are then thawed by 
holding the tube under hot running water (get the hot water flowing 
before you start since ours didn't turn hot till about 5 mins after 
turning the tap on!) and gently shaking the tube. Remove the tube from 
the hot water immediately the suspension has just thawed (and is 
therefore still cold) and transfer immediately into a pre-chilled glass 
homogeniser (on ice) and release the nuclei with 20 gentle strokes with 
the homogeniser's PTFE pestle. The homogenate is then layered onto a 
250µl cusion of pre-chilled 50% sucrose (w/v) in NPB in a 1.4ml 
eppendorf tube and centrifuged at 13,500 rpm in a microcentrifuge (in 
the fridge). The nuclei pellet both to the bottom and up the side of the 
tube so don't throw away the stuff that pellets onto the side walls 
(approx one third to one half way up the tube side). Remove the 
supernatant (containing the cytoplasmic debris etc) as much as possible 
and then resuspend the nuclei very very gently with 100µl of pre-chilled 
NPB (possibly with 0.1-1% BSA to stabilise the nuclei) , leave for 5 
mins on ice and then if necessary dilute the nuclei further to 1ml with 
the same buffer. Take a small aliquot to determine the concentration and 
yield of nuclei using trypan blue, counting chamber and light 
microscope. This will also provide a rough indication of whether any 
whole cells or ruptured cells (nuclei still retaining cytoplasm and 
plasma membrane) are present in the preparation (but only EM, enzyme 
assays and immunofluorescence can confirm this).

This method has always worked very well for me and is very rapid and 
reliable (yield of >40 million nuclei per 50 million cells used). 
However, as already mentioned, you are not using haemopoietic cells, so 
if you want to give this method a try then you may have to alter some 
conditions if it doesn't work as written above:
1) increase or decrease the concentration of Tween 40;
2) use a different detergent (e.g. NP-40 as you already use this);
3) increase/decrease the number of strokes with the PTFE pestle.

Another thing is that if you only want to use approx 5-10 million cells 
you may need to decrease the volume of the initial cell suspension in 
NPB-Tween 40, or try decreasing the concentration of Tween 40.

Hope this helps.
Best wishes,

Kev.





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