a problem of antiboy conjugated with PE - PE bleach out shortly? (Jan/14/2008 )
I just started doing flow cytometry cell sorting. One of the antibodies I used is anti-CD38 conjugated with PE from BD biosciences. The conjugated PE molecule is unstable, it bleached out after the sorting. So I couldn't measure the sorted cells' purity accurately by flow cytometry. Is the bleaching-out a common problem of PE?
Thank you very much!
Do you do your labeling at 4°C?
From what I've heard/ experienced once you expose (excite) the fluor it will bleach out and that's it - you can't get good signal again. I get the impression that it's a one use sort of thing (but don't quote me on that). The tech in the cyto lab that does all of the FISH says because of that reason you have to work fast when using the scope and looking at cells. I've had success with flow sorting (and checked purity after sorting) but that is with GFP expressed protein and not with an antibody.
maybe you are not fixing cell, so cell is capping, and there is no antibody when you try to see the pulity?
can you see the same candition if you use FITC conjugated antibody?
I would also like to say staining at 4C (as Missele said) might help you.
That is a good point. I do all of my staining with cold buffers and at 4C and I also use a staining buffer that has NaN3 (sodium azide) in it. I've read that it's supposed to help prevent the cell from internalizing the antibody after it binds to the cell surface. I'm not sure what the time frame is for internalization but from what you said it seems like you are getting signal lot enough to successfully flow sort.