I need to shorten my MACS cell isolation protocol as much as I can. When I perform positive selection for EpCAM+ve cells, the militenyi manual says to incubate cells with EpCAM-PE antibody first, wash it, then add Anti-PE beads and incubate again. My question is, could I incubate EpCAM-PE with Anti-PE beads first and then do the single incubation? If I can, what incubation times would you recommend? Militenyi says 15min incubation of beads with EpCAM-PE conjugated cells. I work with DispaseII digested mouse lung epithelial cells.
I would not recommend it as there are reasons why you need to add your primary, wash and add secondary (in this case it's your beads).
If you add your primary to secondary, (depending on the cell line you use), steric hindrance may be a problem increasing the difficulty of your Ab binding to your cells, therefore, you may lose some signal. The opposite may also be true, if the antibodies bind to each other forming clumps of Ab, you might be causing more NS binding and getting a lot of false positives.
Whatever protocol is included in your manufacturer's MSDS, I would follow strictly as they have been optimised to ensure the results have reduced non-specific binding.
Hope that helps.
Thank you Darellynn, this makes a lot of sense. I'll stick to the protocol then.
As suggested by Darellyn it would not be a good idea. When both are mixed together you might end up forming enormous complexes which would not be functional. Alternatively if you need a single step you can use the EPCAM microbeads. These are available in Miltenyi.
If you incubate the beads with the primary antibody you can't reduce any unspecific binding by washing between labeling steps with primary and secondary reagent. Also the concentrations ranges will be wrong. If you can get a directly labeled beads (one with the EPCAM on as suggested) I would go for that. If the problem is that you need to stain (e.g. for Flow Cytometry) as well, try staining after the separation - so you would need to check if you can use the same antibody staining twice (one way around would be to stain with a secondary antibody recognizing your EPCAM on the beads).
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