Protocol for staining live cells - Staining live cells for Flow (May/06/2009 )

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?

-Loris-

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

-aimikins-

Loris on May 6 2009, 10:23 AM said:

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?

-illuminated-