Hi,
We use 96-wells a lot for cell assay's and allways find the same effect. We
now call it the edge effect. In our opinion it is caused by the excess
evaporation at the edgess of the plate. The cells are living there under
unvaverable conditions after two days or so (5 days being lethal) because
all concentrations are higher. They even die from the wrong osmotic
pressure.
You can see the effect if you look at the site of the 96-well plate : the
liquid level decreases towards the wells at a1 and a12 etc. The effect is
known to the manufacturers of plates too. They sell low-evap plates which
will last longer in the co2 incubator without refreshing the medium. We
found that obvious alternatives also works : 1) seal a part of the site of
the plate with tape or 2) wrap the plate , or a stack of plates , losely in
saran wrap. Use a thin sort ; it selectively lets co2 pass and retains water
vapour or 3) raise the rel-hum in the incubator drastically and persuate
your peers not to use/open it or 4) insert the 96-wells in a perspex box
with small holes and insert this container in the incubator or 5) do this
locally in each plate by filling the edge wells with a isotonic solution
like phosphate buffered saline. This will tansform the 96-well plate (8*12),
ofcourse , in a 60-well (6*10)
Enjoy your research
Gys de Jongh
Dennis P Smith wrote in message <372EF272.FAC78C27 at lilly.com>...
>We are running a cell based luciferase assay in a 96 well format. To
>test the assay we add a fixed concentration of a known compound that
>gives a positive response to all 96 wells containing transfected cells
>with a Promega luciferase reporter plasmid. Then we incubate the cells
>with the compound in media for 5 hours at 37 degrees, remove the media
>and immediately add the luciferase assay buffer. The luciferase is then
>counted using a Dynex luminometer. Theoretically all the wells should
>have the same relative light units (RLU) but we see an “edge effect”.
>The RLU on the perimeter of the plate is statistically higher then the
>center. Have other people reported is effect? How can is effect be
>avoided?
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This effect is so general and profound. I did the MTT assay for 2, 3 and 4 days. After 2 days, The cells in the edge of the 96-well plate will be dead. So fill the edge with the same medium when you do these kind of experiments.
Yuan-Chin
