weak signal flowcytomtery

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raves
raves's picture
weak signal flowcytomtery

Does anyone have advice regarding how to increase signal intensity for flowcytometry. I currenly use a secondary conjugated to an Alex488

marcus muench
marcus muench's picture
There are a number of things

There are a number of things you can do.

First, although AlexaFluor 488 is not a bad dye, PE may be a better choice when working with weak signals.

Make sure your cells are healthy and try and use propidium iodide or some other dye to distinguish live from dead cells. Dead cells tend to be sticky and have a higher background staining, thus decreasing your signal to noise ratio.

Try blocking non-specific binding. If your using a secondary Ab you have to make sure that your not blocking with something recognized by that antibody. For instance, mouse serum is not a good idea if your primary is mouse and your secondary is goat anti-mouse. In such a case you could use human IgG, some other serum or even FBS can help depending on what kind of cells your working with.

Boost your voltage signals as much as possible. IF your signal is weak, then there is no point squeezing your events below the first decade on the graph. Pull out your negative controls by increasing the voltages, which may help distinguish positive from negative signals.

Jason King
Jason King's picture
I find the Alexa-647 really

I find the Alexa-647 really useful. Dead cells and macrophages often have an autofluorescence associated with them which could be a problem with a 488 detection of weak signals, but the 647 is miles away from any autofluorescence signals.

I'm lucky that I can use a primary Alexa647 conjugated Ab, but you might need the amplification effect of the secondary.