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Rat IgG-Biotin-StrepAPC problems


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#1 LCameronLab

LCameronLab

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Posted 03 July 2012 - 04:11 PM

Hi Everyone,

I'm having some very frustrating problems with flow cytometry. I stain human Th2 cells with Mouse IgG antibodies to CD4, CD8 to characterize their "Th2-ness". I also stain for CRTh2 which is quite a specific marker for Th2 cells. Recently I have been having problems with the CRTh2 staining, but all other stains are going fine. The differences between the CRTh2 stain and the others are that it is a Rat IgG conjugated to biotin. I then add Streptavadin-APC. So it is not a direct stain where the others are (Mouse IgG FITC for CD4 and Mouse IgG PE for CD8).
I am only having trouble with the indirectly biotinylated stain for CRTh2. Has anyone used such flow stains before that might be able to troubleshoot?
I have some ideas about the problems:
I keep all tubes on ice almost all the time while doing protocol steps. When they incubate they are in the fridge at 4C. The only exception is a blocking step at the beginning that is incubated at RT. Other people in my lab who get better results don't have their flow tubes on ice all the time. I'm wondering if even the few minutes they do a protocol step off ice might be making a difference in antibody/biotin-Strep binding kinetics? Is it possible that I am being overly zealous by keeping the cells at 4C all the time?
Also others just use pipet to mix, i use pipet and flick to resuspend pellets and mix with antibodies etc. Could I be over doing resuspension/mixing?
Does anyone know if there are specific things commonly done in flow that can cause cells to internalize receptor?
It's really frustrating me because these are the ONLY things I do different and they are things that were recommended to do. I do them because I was told it is better to do them. Anyways, I need to find out if anyone thinks this could be the issue because when I bring it up here nobody seems to think it could possibly be the problem. But, I'm out of ideas anyways. Anything anyone can contribute would be greatly appreciated!




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