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> I have a user who has been assessing viability of neural cells using
> Molecular Probes Eukolight kit which uses Calcein AM for live cell
> measurement and Ethidium homodimer for dead cell measurement. My
> question is this: Since ethidium staining is related to leakage into
> the cell as a result of altered membrane integrity and calcein is
> related to cytoplasmic esterase activity, what are the kinetics with
> respect to the sequence of events? Is it possible for cells to stain
> with ethidium while still maintaining esterase activity; hence stain
> with both dyes, i.e. damaged but not dead? Since these samples were
> run on a FACscan which cannot compensateFL1 and FL3 and there is
> significant spectral overlap between these dyes it was not possible
> to look at the dual color staining by flow. However using
> fluorescence microscopy we definitely saw dual staining. Could it be
> that the cells were damaged but still had some esterase activity? I
> would appreciate any responses from people who have worked with this
> kit before.
I can't answer the actual question on esterase activity after cell
damage from first hand experience, but I can tell you how to do the
compensation so that you might answer it yourself on your FACScan:
If Ethidium fluorescence leaks into FL1 then it must leak more into
FL2. Set FL2-%FL3 to 0.0 to allow that leakage into FL2. You can
then remove Ethidium fluorescence from FL1 by adjusting FL1-%FL2.
Similarly, if Calcein fluorescence leaks into FL3, then it must leak
more into FL2. Set FL2-%FL1 to 0.0 to allow leakage into FL2. You can then
remove calcein fluorescence from FL3 by adjusting FL3-%FL2.
\ / < Flow Systems Laboratory
Frank Battye \__/ <<<<< The Walter & Eliza Hall Institute
----------!!<<<<<<<< Voice: 61_3_9345 2540
/!!\ <<<<< Fax: 61_3_9347 0852
o !! \ < IN:: "facs_copy@wehi.edu.au"
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