I am trying to sort cells based on a biotnylated-protein tetramer. After biotinylation I have to option to bind my protein to S-APC or S-PE beads in the protocol and wanted to know if there are any fundamental differences that would affect my flow cytometry?
Hi Justin,
I don't expect a fundamental difference in S-APC or S-PE as both dye conjugates are bright. Streptavidin itself is also a tetramer, and depending on the expression of your epitope which you described as a tetramer, you would probably have very strong signal in either case. Are you co-staining your cells with other colors (or are your cells already fluorescent)? If so, that would probably be the biggest determinant of APC v. PE.
Thanks,
Barry
As Barry said both PE and APC are bright non-tandem fluorophores that work well for tetramer staining. The only issues you might encounter are if you have other fluorophores that might degrade into these channels. For example, PE-Cy7, PE-Cy5, PE-Cy5.5 are all PE tandems (PE core bound to another component that gives them a unique fluorescence) that can degrade (fixation, vortexing, UV light exposure, etc) into the PE core plus the tandem giving you a false PE signal. APC-Cy7 and APC have a similar issue. Depending on your panel you may try to minimize the amount of false-positive signal that could occur from tandem degradation (i.e., use APC if you have a lot of PE tandems present). A second consideration is your threshold of detection for your tetramer channel. For example, you will lose some of your APC signal if you use A700 due to spectral overlap and compensation. The best channel in your case will probably be a mixture of minimizing tandem degradation and loss of signal occurring from compensation.
Hi Justin,
I did work with both PE and APC to sort Env-specific memory B cells, and both work nicely, as the others said the main corners are the other colors in the panel. As Brett mentioned you will lose some of the APC signal if you use A700, as well as PE if you use PECF594.
What we did in the lab was to first stain with the protein-S-PE/APC incubate for 20min at 4°C, wash 3x and then do the surface stain. We also titrated the amount of protein to be use and all the antibodies in the stain
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