gotta get those #$@&* cells to STICK!!!

john.hines at yale.edu john.hines at yale.edu
Tue Feb 26 18:06:52 EST 2002


As you can probably tell from my subject line, I am having some profound
difficulties getting my cells to stick to tissue culture dishes.  This is
a problem, since I want to do IHC on them.

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.

I've tried overloading the dishes in anticipation of some cell loss, as
well as being *very* gentle with the medium changes, PBS washes, etc. 
Still not so good.

I am planning on trying out a few different dish coatings (fibronectin,
poly-L-lysine, collagen, etc.) to see if one of them retains the cells for
longer.  This whole glitch is was somewhat unexpected, since gelatin seems
to be the coating used in the literature; and since it worked fine for a
little while in my hands -- just didn't work fine long enough.


Does anyone else have any suggestions?  I wonder if I am missing out on
something simple?


John

john.hines at yale.edu




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