In Flow Cytometry we measure light, frequently fluorescent light. A fundamental question is how to pick the right fluorochromes for your experiment.
In planning of an experiment, the combination of fluorochromes with targets is critical especially when investigating cells occurring at low frequency or when utilizing multi-parameter flow cytometry. Two points are of particular importance when deciding which fluorochromes to use. First, not all fluorochromes are made equal; some are brighter than others. Second, the final decision of which fluorochrome to use maybe dependent upon; the number of lasers in the flow cytometer, confidence in setting the compensation parameter for the second laser and/or which antibody can you find in a catalog! A good method is to generally stain the least frequently expressed epitope with the brightest fluorochrome and visa versa. Brightness depends not only on the inherent qualities of the fluorochrome, but also on the detectors/filter sets of the equipment that will detect the light, so the same fluorochromes might have different order using different machines. A general guideline to the relative emission wavelengths of each fluorochrome is shown below.
Brightest BV421, PE, PE-Cy7, PE-Cy5, BV 605, APC > APC-Cy7>Alexa Fluor 647, , BV711, BV785>Alexa Fluor 700 >BV510, BV 650, FITC, Alexa Fluor 488, Pacific Blue, BV570 Least bright
Picking the fluorochromes that work well together in a multi color experiment is very important. We will help you with this task when discussing your experimental plan, but if you would like to be proactive, here are some ideas how the various fluorochromes might work for you.
One can certainly pick all sort of fluorochromes with overlapping emission spectra. There is a procedure, compensation, to make up for these interferences in terms of medium fluorescence values, however compensation does not make up for one fluorochrome beating up on detectors other than its own. So, it is beneficial to pick fluorochromes, if possible, that do not interfere with each other’s detection. The easiest way to go about this is to look at any of the many fluorescent spectra viewers on line.
The fluorochromes one can use in an experiment are limited by the availability of reagents as well as the instrumentation. The following chart is designed to give you an idea about commonly used reagents in 6, 8 and 10 color experiments.
|6 Color||8 Color||10 Color|
|FITC or Alexa 488||FITC or Alexa 488||FITC or Alexa 488|
|APC or Alexa 647||APC or Alexa 647||APC or Alexa 647|
|-||-||Alexa 680 or 700|