gotta get my $%#&* cells to STICK!!

Warren Gallin wgallin at gpu.srv.ualberta.ca
Tue Feb 26 19:17:52 EST 2002


John,
	One possibility is that the matrix adhesion is exquisitely calcium
sensitive (those darn integrins).  Most PBS formulations do not have
calcium and magnesium added, and the phosphate tends to complex with any
trace amounts.
	What I do if this is looking to be an issue is to add CaCl2 and MgCl2
to 0.5 mM final concentration in an aliquot of PBS that is being used
for washing.  It may get a little opalescent (Calcium phosphate is
poorly soluble in water, thank goodness) and if you are worried about
the trace solids you can ultrafilter it.
	The other thing I have found with some cell lines (No information
specifically on myotubes) is that at low temperature they release
quickly from the substrate, so for those cell lines that are sensitive I
use prewarmed solutions when rinsing, at least until they have been fixed.
	Of course, your mileage may vary

Warren Gallin

john.hines at yale.edu wrote:
 
> They are a muscle(myoblast)-derived cell line that has been differentiated
> to form multinucleate myotubules.  I have been trypsinizing the myotubules
> and then filtering them through microsieves in order to get rid of any
> remaining myoblasts.  Then I replate them onto gelatin-coated dishes.
> They stick immediately and then, with time (3 days), they spread out
> again.  Good.
> 
> Of course, with the first PBS rinse, they come right off of the bottom.
> Not so good.




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