Compensation properties of distinct amine-reactive derivatives of fluorescein

Fluorescein is one of the most commonly used fluorophores in flow cytometry. There are a number of distinct amino-group reactive derivatives of fluorescein, with "FITC" being only one among them (the "ITC" in FITC stands for "isothiocyanate"). Although these dyes are structurally similar (and antibodies labeled with any one of them can be used with confidence), they have slightly different emission spectra, and therefore their compensation properties versus the FL2 channel on most flow cytometers are slightly different. As a consequence, it is not a good idea to set instrument compensations with a DTAF-labeled antibody when you are using FITC- or NHS-fluorescein-labeled antibodies in your subsequent experiments.

Unfortunately, it appears as if some manufacturers can be sloppy with labeling of antibodies as "FITC" labeled when in fact, sometimes they are labeled with DTAF or NHS-fluorescein. BD/Pharmingen appear to use FITC, Immunotech often uses DTAF (at least they used to), Molecular Probes often uses NHS-fluorescein (e.g. their streptavidin-fluorescein conjugate, which is not well-labeled on their website), and I don't know what Caltag uses.

Amine-Reactive Derivatives of Fluorescein
FITC. Available from Molecular Probes, Pierce, and other suppliers	DTAF. Available from Molecular Probes, Pierce, and other suppliers	NHS-Fluorescein. Available from Molecular Probes, Pierce, and other suppliers