I have a fluorescent probe that I'd like to test using flow cytometry, so that I can quantify its relative binding to different cell types. Unfortunately, the flow cytometer at my institution does not have a laser that reaches its recommended excitation wavelength. It does have a laser which overlaps its excitation spectrum, but only at a point which corresponds to 13% of the maximum excitation potential.
If I use this machine on samples treated with this probe is it possible that I can pick it up if the signal is strong enough, or am I risking inaccurate results?
If it only reaches 13% of your maximum excitation potential, you won't get a good excitation signal, you are risking incaccurate results.
Dear Isabel
Sometimes a fluorescence molecule has several peaks in its excitation spectrum, which only the highest peak is introduced by the manufacturer (Of course, this issue may be considered a defect in the case of commercial probes). Nevertheless, you can first dilute a very small amount of your fluorescence probe in a suitable buffer used in flowcytometry and take a "spectrum" with a fluorescence reader. One of the smaller excitation peaks may be the one your flow cytometry device has a laser for.
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