Thankyou to all who replied to my query regarding correlation (or lack of) between FACS and trypan blue exclusion. In this series of experiments we are comparing a panel of reagents for ability to deliver reporter constructs into a variety of cell types. For several boring reasons we decided not to use GFP (our usual choice) but to use bgal. We have not been happy with accurate quantitation of bgal transfection using FDG and FACS so are using chemiluminescence detection. However, we also need to quantitate the viability as there are considerable differences in toxicity of different reagents looking at the cultures. As each experiment involves large number of samples we thought that a quick way to quantitate this would be to determine percentage viable by FACS. We now know better. Manual counts with trypan blue are loo labour-intensive on a large scale. So we have now decided to determine the total number of viable cells (rather than the percentage as before) per sample. We plan to do this by spiking each sample with beads of known concentration prior to FACS. Comments on pitfalls of this approach would be appreciated. In future such experiments I would like to use a fluorometric plate reader for the bgal and viability assays, but its not on budget until next year... Thanks, Michelle JJR, Sydney, Australia
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