Cell numbers versus confluency - (Oct/23/2012 )

Hi all! I have a question for you. I am looking at relative uptake of a compound between two cell types (microglia and astrocytes). Ideally, an equal number of cells would be treated at equal confluency, however astrocytes are quite a bit bigger than microglia. Therefore, if each plate is seeded with one million cells of either microglia or astrocytes, the astrocyte plate is going to appear much more confluent than the microglial plate. Likewise, an 80% confluent plate will have many more microglia than astrocytes.

Which situation would you consider preferable? Equal confluency (ie, both treated at 80% confluency) or equal number of cells (ie, 1 million cells treated regardless of confluency)?
Thanks!

Could you plate the astrocytes in a larger plate, so that both cell number and confluence would be the same? Then you could adjust the volume of your treatment to account for the greater plate volume.

Another way would be to keep confluence the same and normalize for cell number at the end, i.e., express uptake in the form of umoles/million cells or something like that.

Personally, I'd go for identical cell number. It is the only objective measure. You could get 10 people to look at a plate of cells and they would not all necessarily agree that it is 80% confluent, so I think this is a very poor measure.

If you are worried that the state of confluence the cells have reached will have an effect on compound uptake, you could try examining uptake for each cell line at different densities to work out if this is going to be a problem.