In Flow Cytometry we measure light, frequently fluorescent light.  If you are not familiar with issues concerning fluorescence and how those relate to biological work, there are some good tutorials on the topic on the Invitrogene website here.

The Invitrogen tutorial gives a good idea about the nature of fluorescent dyes.  There are two more items worth mentioning.  One is compensation, which is well covered in tutorials under the "Flow Cytometry" tab.  The other one is brightness of fluorochromes, in terms of which one to pick for which parameter you are interested in. In the preparation and planning of an experiment, the combination of fluorochromes per acquisition 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 e.g. PE and APC both emit a higher wavelength when excited and so stain with a higher relative fluorescent intensity. 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 you can find at the time! A good method is to generally stain the least frequent cells with the brightest fluorochrome and visa versa. Brightness depends not only on the inherent qualities of the fruorochrome, 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  PE, PE-Cy7, PE-Cy5, APC > APC-Cy7>Alexa Fluor 647, Alexa Fluor 700 >FITC, Pacific Blue, Alexa Fluor 488 Least bright

So, there you are.  The only commercially available antibody (and it is a great antibody) conjugate to your rare population is FITC instead of APC.  Is there anythithing that can be done to help this situation, you ask.  Milteny Biotec thinks, yes. FITCenhancer.pdf  If your staining of APC or PE is not strong enough, they have a kit to enhance those, too.

 

Regarding the brightness there are more issues that make the question tricky.  What if dye A is brighter then dye B if excited at the optimal excitation wavelength, but the one in the flow cytometer is closer to the optimum for B than for A?  You are using the dyes for flow and for immunocytochemistry, A is brighter than B, but B is more photo-stabile, and you are likely to spend some time scanning the slide before taking a picture.  Which one will be brighter at the time of talking a picture?  You can read more on this topic by Mario Roederer or the BD 2006 tech resource page BDISTechResources2006.pdf.

 

Another important question is which fluorochromes can be used with our equipment.  The following tables should be a good guide in this regard for the basic configuration of the Canto II and the LSR II.  The most frequently used fluorochromes are highlighted, the ones that can be detected with the particular detector in a sub-optimal back up scenario are in parenthesis.

 

 

 

 

While the list above is quite large, but by all means is not a definitive one.  If you have something else on your mind, discuss it with the Flow Lab.