dissociation question

Richard Kerr kerrr at CRYPTIC.RCH.UNIMELB.EDU.AU
Thu Nov 14 19:53:10 EST 1996


At 17:40 14/11/96 GMT, you wrote:
>What is a good enzyme to use in dissociation of DRGs if I want to
>remove extracellular matrix?  I've been trying all sorts of
>collagenase, typsin and dispase combinations and I can't shake the
>last few glia cells adhering to the DRG neuron somata.
>
>Anyone have any suggestions?
>
>je
>
Hello john,
you seemed to have covered all the good enzymes for acieving your goal.  i
have a few thoughts which may be useful to you.

1.      do you run your dissociation digests in Calcium-free or depleted
conditions (ie: 2-5 mM EDTA) ? the answer to this is probably "yes" but you
don't mention it in your post.

2.      have you tried testicular hyaluronidase ?
It's got good  digestive properties against HA and CSPG and because its from
bulls bollocks, you get a lot of proteases as well.  The proteases are
heterogenous but don't seem to harm cells in the same way as pancreatin
preps and high levels of trypsin.

3.       is it really that crucial that you get rid of the 'few ' glia that
remain ?  if it is then try a longer gentler digest but put your
dissociation prep. on some kind of roller/rocking platform..this often does
the trick.

Hope this helps.
R 
Richard Kerr.
The Murdoch Institute,
R.C.H. Flemington Rd, Parkville, 3052,
AUSTRALIA.
kerrr at cryptic.rch.unimelb.edu.au
Phone (61) 3 9345 5045.




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