Wherever possible you should use fluorescent dyes and proteins with as little spillover as possible between them. This will minimise the data spread introduced by these spillover effects and, in turn, maximise the sensitivity of your assay and accuracy of your measurements.

To achieve minimal fluorescence spillover:

• Use fluorochromes which are excited by different lasers (eg. on the four laser LSRII you could excite FITC/A488/GFP with the blue laser, PE with the yellow/green laser, APC/A647 with the red laser and Pacific Blue with the violet laser).
   
• Use fluorochrome combinations with as large a difference in emission wavelength as possible (eg. PE-Cy5.5 or PE-Cy7 could comfortably be added to the list of dyes above).

To assist with choosing well separated fluorochromes, both BD and Invitrogen have spectrum viewers available on their web sites (see links below). You can use these to match your fluorochromes to the lasers and filters in the flow cytometer you plan to use, as well as visualising the amount of spillover to expect.

If you are spoilt for choice and only need a few colours in your assay, go for the brightest fluorochrome you can (typically the large protein dyes, i.e. PE and APC). In descending order of brightness, here is a list of recommended fluorochromes:

• PE
• APC/Alexa647
• PE-Cy5.5/PE-Alexa700/PE-Cy7**
• FITC/Alexa488
• Pacific Blue.

**Whilst PE-Cy7 can be an exceptionally bright and useful fluorochrome, it is highly susceptible to oxidation (which will cause it to fluoresce increasing at the PE emission wavelength rather than the Cy7 emission wavelength). It must therefore be treated extremely well, minimising the time it is out of the fridge and exposed to light.

This list is by no means exhaustive, however - feel free to ask a FlowCore staff member for more information about fluorochrome choice.

Even if you are exciting fluorochromes with individual spatially separated lasers, it is generally not a good idea to use fluorochrome combinations with similar or identical emission wavelengths (remembering to consider the viability dye as well). Some examples include:

• Pacific Blue and DAPI
• RFP and PI
• APC/Alexa647 and PE-Cy5
• PE and Alexa568/594
• APC and APC-Cy7 (even though these are quite well separated, in practise we have found it a poor combination).

If users are your choice of fluorochrome combinations, please ask a FlowCore staff member for advice.