Michael S. Straka
mike.straka at uchsc.edu
Fri Mar 22 12:17:10 EST 1996
In article <Pine.HPP.3.91.960321153016.22171A-100000 at bluejay.creighton.edu>, Steven Vokal <vokal at creighton.edu> says:
> I am doing alot of cell culture work with 24 and 96 well culture
>plates. I seem to have the problem of not being able to put the same
>number of cells in each plate accurately. Also it seems the cells grow
>differently from well to well. Can someone suggest a way to solve my
> Steve Vokal
> Dept of Surgery
> Creighton University
> Omaha, NE
I have worked mostly w/ human and rat hepatocytes/transformants,
and CHO and other fibroblasts. You don't say what cell type you're
working w/ but I'll venture a few hints anyway.
I assume you collagenase the cells from your farm and do a count
prior to plating. In my experience, it's easier to work w/ a relatively
dilute suspension, no more than about 1 - 2 x 10E6 cells/ml; it allows
for more reproducible pipetting. Make sure you swirl the suspension
regularly, say after pipetting every 3 or 5 wells, something like that.
You also don't mention your experimental design; for a short term expt
(12-48 h), you can seed them a little heavier - up to about 1.5
million/well (24-well); for a longer term expt, I recommend lighter
seeding to avoid overgrowth. Growth of many cell types is affected
by plating density - your problem of differential growth may resolve
itself once you settle on an appropriate seeding density. Thus, you
should try different seeding densities to see which one gives you the
growth you want. I also assume you do a protein or DNA msmt as a
denominator; differences of about 20 - 30% between wells (depending
upon treatment) are acceptable. I haven't done much w/ 96-well plates,
so you're on your own there.
Good luck, and feel free to email me if you have other questions.
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