|How are you going to evaluate your samples; flow cytometer or fluorescence microscope. And what filters do these contain.|
|What is the expression level of the target.|
|What combination of fluorochromes do I want to use and do I want a single, double or multi...... staining.|
For targets with a low expression a fluorochrome with a high
'energy', like phycoerythrin (PE), is advised. At least
when a flow cytometer is being used. For use in a fluorescence
microscope PE is not a wise choice. The signal will be gone before the microscope
can be focused and making a photo will be very difficult. PE is one of the brightest
fluorochromes today. One molecule of this protein has 25 'fluor-groups'. Which
makes it powerful and extremely useful in the flow cytometer.
It is also wise to think of the possible combinations that have to be made (now and in the future). In the case of cytokine staining most of the Abs available are conjugated to FITC, PE or allophycocyanine (APC). When a flow cytometer from BD is used this means channel 1, 2 and 4 have the possibility to detect cytokines (intracellular proteins) and are left with the third channel to use for a surface or activation marker. The FITC conjugated ab is used for the most abundant produced cytokine and the PE and/or APC for the cytokines that will be harder to detect.
But in the end it will be the method of detection that is the factor that determines what fluorochrome has to be used. Using Texas Red in a sample and coming to the conclusion the fluorescence microscope doesn't have the right emission filter makes the sample ''worthless''.
Although we did our best we can
not be held responsible for any mistakes or typing errors.