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Protocol for staining live cells


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#1 Loris

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Posted 06 May 2009 - 09:23 AM

Hi everyone,

Does anyone have a protocol for staining live cells with antibodies against antigens presented on the surface?

Also, on a side note, someone told me that formaldehyde can quenche fluorescence of some fluorophores. Is this true?

And something else, I like to use propidium iodide for staining DNA (mainly for cell cycle analysis). I get weird results if I formaldehyde fix. Does anyone know why?

:)

#2 aimikins

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Posted 06 May 2009 - 12:24 PM

I would say it's not that easy? it depends on the density of expression of your receptors; how clean is your antiboy; what type of cells?

for general protocols, BD Pharmingen's website is very informative and can give you a place to start, based on your cell type and what you are trying to find. they also have great antibodies :)
"it is a miracle that curiosity survives formal education" -A.E.

#3 illuminated

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Posted 08 June 2009 - 06:54 AM

And something else, I like to use propidium iodide for staining DNA (mainly for cell cycle analysis). I get weird results if I formaldehyde fix. Does anyone know why?

Why don't you fix with ethanol? (Para-)formaldehyde has an intrinsic fluorescence, and beside that you cannot store the fixed cells for long (e.g. for later analysis). In general, a FACS-expert told me that PI staining results (for cell cycle analysis) of PFA-fixed cells are less reliable then those of ethanol-fixed cells.

For which surface antigens to you want to stain?




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