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Live/dead staining


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3 replies to this topic

#1 canotto

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Posted 10 October 2009 - 04:19 AM

I've read that there are a number of molecules that enter live cells and react with intracellular esterases to yeld a fluorescent product wich is retained as long as the plasma membrane remains intact, such as fluorescein diacetate ( FDA), carboxyfluorescein diacetate (CFDA) and its acetoxymethyl ester (CFDA-AM). I don't have any of these products in the lab, but I have 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE). Do you know if this probe can be used also fot the tracking of living cells?
What I wanted to do is a two-color analysis with Propidium Iodide and a dye for living cells

#2 bachai

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Posted 11 October 2009 - 07:23 PM

What experiments you want to do?
a) For seeing (live) cell activities in vitro over a period of time (e.g. proliferation), or tracing them after being injected in vivo, CFSE is probably OK. However as a lipid-soluble dye this is not specific to live cells. Actually those which are brightest on CFSE would be the dead cells which are not capable of dividing and therefore diluting the dye out during your experiment.
:P For a two-color FACS to see what cells are dead and what are live, use Annexin-V plus PI. Cells single-positive for Annexin-V are early apoptotic, double-positive are late apoptotic (necrotic), and live cells must be negative on both.

#3 canotto

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Posted 16 October 2009 - 05:13 AM

What experiments you want to do?
a) For seeing (live) cell activities in vitro over a period of time (e.g. proliferation), or tracing them after being injected in vivo, CFSE is probably OK. However as a lipid-soluble dye this is not specific to live cells. Actually those which are brightest on CFSE would be the dead cells which are not capable of dividing and therefore diluting the dye out during your experiment.
:wacko: For a two-color FACS to see what cells are dead and what are live, use Annexin-V plus PI. Cells single-positive for Annexin-V are early apoptotic, double-positive are late apoptotic (necrotic), and live cells must be negative on both.



I wanted to do a drug-induced cytotoxicity assay ( see King MA et al. J Immunol Methods 2000)

#4 sahana001

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Posted 18 October 2009 - 09:35 AM

hi !
its really a informative post...
thanks for your information ......


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