The formaldehyde-fluorescamine method. An investigation of reaction mechanisms and optimization of the method.

Formaldehyde reacts with primary amino groups to derivatives which are unable to react with the fluorogenic primary amino group probe, fluorescamine. Paradoxically, however, certain specific cell systems continue to display strong fluorescamine-induced fluorescence after formaldehyde pretreatment. Among such formaldehyde-fluorescamine (FF) positive cell systems are certain peptide- and protein-secreting cells as well as all hitherto investigated types of cancer cells. We have now optimized the cytochemical FF method by using microfluorometry in combination with systematically varied reaction conditions. In addition, the quantitative data indicate that in FF positive cells, formaldehyde pretreatment causes a paradoxical increase in the fluorescence yield with fluorescamine. This has tentatively been ascribed to quenching phenomena, associated with closely spaced primary amino groups. Work with alternative fluorogenic amino group probes (MDPF and OPT) show that these display the same spectrum of tissue selectivity as fluorescamine, but that the latter remains the reagent of choice for the cytochemical FF reaction.