Does anyone have experience with Calcein AM/EtD-1staining for cytotoxicity assay - (Oct/30/2014 )

Hey!

I'm new to this forum and to be honestnew to forums in general, som please be kind if I'm reposting or post this where it doesn't belong.

Anyway, I'm having some trouble with finding the optimum concentration of calcein AM and EtD-1 for a cytotoxicity assay. I'm using the Live/dead assay from Molecular probes and I would like to do the assay in a 96 well microplate. But while I now have the data for several different concentrations for both dyes as well as reading at different timepoints, I'm not entirely sure how to analyze it.

My original thought was to plot using the concentrations of the EtD-1 on the x axis and the fluorescence on the y axis, then having the timepoints as different data series in the graph. But my supervisor wants to use the concentrations as data series and the time on the x axis. Yet she cannot explain to me why this would be more correct. She also tells me that there is interference between the two dyes, which seems unlikely as it is a kit we are using.  Does anyone have experience with using these dyes or the actual kit as a microplate assay?

Thanks, and best regards!

Concentration as the data series over time (X) would (presumably) give you a number of independent bar graphs that (or lines depending on how you plot) that could allow you to assess the relationship between death and time, in essence allowing you to get a minimal dose for killing the cells within the time period. Your way would work too, but would probably be less intuitive for most people (perhaps).

Thanks for your reply bob1!

I agree that this will be the best way to interpret data once I've got the assay up and running. However I think I might have been a little unclear in my post, as my trouble is with the initial optimization of the the dyes (calcein and EtD-1). I need to find the saturating concentration and incubation time of EtD-1, and then a concentration of Calcein that gives the best ratio between the signals for live and dead cells.