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BaF3 cell culture in 96 well plates

Ba/F3 well plate attachment coating suspension cells

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#1 turboprince

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Posted 07 December 2012 - 11:43 PM

Hi!

We are new to the forum and seriously need some help here!!

We are working with Ba/F3 cells (mouse pro-B-cell line the growth/proliferation of which depends on Interleukin 3; these are suspension cells) and regular cell culture works fine (cells look like in papers, withdrawal of interleukin 3 leads to cell death).

Problems start as a soon as we are trying to grow the cells in well plates: We tried about ten different plates from about six different manufacturers that had all kinds of surface properties and that were 24, 48 or 98 well and round or flat bottom. In all cases we observe the same thing: The cells sink to the bottom of the well, stick to the bottom and die... at the same time, the culture in the regular flask we use to propagate the cells is fine, some cells sit at the bottom but they are quite loose, which in the wells all cells sink and die.

We tried BSA coating (0.5% for 2days at 37degC) and coating with "HEMA" (poly 2-hydroxyethyl methacrylate, 20mg/ml, following the establised protocols) and no change...

This is really getting a bit frustrating so any help would be GREATLY appreciated...

MANY thanks in advance!
Harold

#2 bob1

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Posted 08 December 2012 - 12:33 PM

Welcome to the forums.

What sort of density are you seeding the cells at in the wells vs in the flasks?

Ratio of medium : cells?

That sort of behaviour (the sticking to the bottom) is often a sign of differentiation in these sorts of lines IIRC, which is what makes me think it is something to do with seeding density.

#3 turboprince

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Posted 08 December 2012 - 11:37 PM

Welcome to the forums.

What sort of density are you seeding the cells at in the wells vs in the flasks?

Ratio of medium : cells?

That sort of behaviour (the sticking to the bottom) is often a sign of differentiation in these sorts of lines IIRC, which is what makes me think it is something to do with seeding density.



Hi Bob.

Thanks for your message.

Seeding density is the same as in the bottles and range from about 5 X 10^4 to 5 X 10^5 cells/ml, which is what we were told keeps the cells from developing e.g. IL3-independent growth...

Thanks again.
Harold





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