cell death in plates

David Rusnak rusnak~dw at glaxo.com
Mon Oct 28 11:40:26 EST 1996

Thomas Kreuzer wrote:
> Hello fellow netters,
> I have been faced with a problem recently , for which I can find no solution
> altough it may be trivial.
> I seed HeLa - cells on standard 6-well cell culture plates (Costar) in 1 ml
> DMEM plus 5 % FCS , let them attach to the bottom overnight and then change
> the medium according to the tests I want to perform afterwards. I have noticed
> for some time that the cells in certain wells show a different behaviour than
> the others. For example: If I number the wells from left to right 1 to 3 in
> the upper row and 4 to 6 in the lower , the cells in number 4 are always more
> difficult to get off the bottom of the well with trypsin. First I thought it
> might be a handling problem during the tests , but last time I decided to let
> the cells grow for one additional day on the plate before the test , so I did
> not change the medium after the first night , but left it for further 24 hours.
> When I looked through the microscope afterwards , I noticed that the cells in
> wells 1,2,3 and 5 had grown well , while in number 4 and 6 most of the cells
> were dead , altough an equal amount of cells had been attached to the bottom
> after the first night in all wells. The conditions were the same for all 6
> wells.
> So my question is: has anybody noticed a similar effect or has some
> suggestions why this might happen ?
> Thanks in advance,
> Tom
> -----------------------------------------------
> Tom Kreuzer
> Institute for Biochemistry,K-F-University Graz,Halbaerthgasse 5,A-8010 Graz,Austria
> thomas.kreuzer at kfunigraz.ac.at

In multi-well dishes I find the the outside wells lose moisture due to 
evaporation faster than wells on the inside of the plate--even in a  
humidified incubator.  This will be much more evident in plates with 
distinct "inside" and "outside" rows (12 or more wells).  The greater the 
evaporation, the greater the change in salt concentration in a given 
well.  This can affect cell growth.

In a 6-well dish, the four corner wells get the same amount of exposure 
to air.  The center wells will be slightly protected from evaporation by 
the corner wells.  I suspect that wells 4 and 6 are losing more moisture. 
I realize that if my theory is correct, one would expect wells 1 and 3 to 
respond like 4 and 6.  Do you put your plate in the incubator in the same 
orientation each time (or reference your numbering system by the way the 
plate is placed in the incubator)?  I am thinking the maybe you have more 
air flow or something to cause more evaporation on the side of the dish 
that wells 4 and 6 are located.

Make sure your incubator has plenty of water.  Seed your cells in more 
media if possible.  You are using 1 ml and it doesn't take long to 
evaporate enough water from the well to drastically effect salt 
concentrations.  I usually use 3 ml in a 6-well dish--2 would be a 
minimum.  If you require smaller volumes to save a precious growth factor 
or reagent, keep your cells in 3ml until you need to dose, and dose for 
as short of a period as possible. Try putting your plates as far to the 
back of the incubator as you can to protect from dry air (unless, of 
course, the incubator has a vent hole back there).



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