#Bio rad marker protein how to
The following guide provides advice on how to measure antigen density and lists the antigen density of some common markers. Knowing the antigen density will help rank markers and pair them to similarly ranked fluorophores. However it becomes more complicated as you increase the number of fluorophores in your panels, especially if the antigen density is unknown or may change with cell subsets or activation state. It is possible for bright fluorophores to be paired with highly expressed antigens, but better to avoid pairing dim fluorophores with low abundance antigens.Ĭare should be taken however, as fluorophore brightness can have both positive and negative effects.Ī bright fluorophore will give better resolution, but have greater spread when compensated, and if placed on an abundant antigen, the increased spillover into adjacent channels may mask the signal from dimmer fluorophores.īuild multicolor flow cytometry panels in just a few simple stepsįluorophore choice is relatively easy when only a few markers are required.
Careful choice of fluorophores will help with the resolution of your cell populations. Pacific Blue) with highly expressed markers. PE) with low expressing markers and dimmer fluorophores (e.g. As a general rule you should pair bright fluorophores (e.g. The number of antigens, or target molecules that a cell carries directly correlates to the intensity of the positive population and will determine the optimal fluorophore you should use for each marker. Tutorials for building multicolor flow cytometry panels always highlight the importance of antigen density - But why is it so important?
Antigen Density of Common Human and Murine Markers
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