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problem with GFP quenching!


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2 replies to this topic

#1 vitalgene

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Posted 13 November 2009 - 11:28 AM

Hi everyone,
its been a frustating weeek for me, I have a cell population which are nearly 70% positive for GFP, upon fixing using the BD cytofix, and there by washing with BDcytoperm, I lose the GFP content to 30% or less, makeing me difficult to stain with some antibody hence hindering the multiplex reaction, Really dont know how to tackle this issue, one way it quench the GFP completely but how?, or are there suitable ways to fix without detoriating the GFP content! please guide with your useful suggestions. Thanks in advance.

#2 miBunny

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Posted 13 November 2009 - 06:58 PM

GFP will definitely leak out of cells that are not fixed with at least 4% PFA (we have tested this with 0.5-4% PFA). I have not tried the BD cytoperm/cytofix with GFP but my protocol is to fix cells with 4% PFA and then perm with 0.1% saponin in facs wash. All cells remain GFP positive

GFP will be quenched by methanol and changes in pH.

#3 vitalgene

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Posted 16 November 2009 - 06:17 AM

GFP will definitely leak out of cells that are not fixed with at least 4% PFA (we have tested this with 0.5-4% PFA). I have not tried the BD cytoperm/cytofix with GFP but my protocol is to fix cells with 4% PFA and then perm with 0.1% saponin in facs wash. All cells remain GFP positive

GFP will be quenched by methanol and changes in pH.



Thank you miBunny, I m using the method from you.
thanks again




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