Calman Prussin asks: > >Does anyone know of a metachromatic fluroescent dye that would stain >basophils or mast cells in a similar way to Alcian blue or toluidine blue? >Any that would work on a 488 nm argon laser? Thanks. The best metachromatic dye I know of for basophils and mast cells is basic orange 21, which works both in absorption (purplish granules) and fluorescence. However, since the orthochromatic excitation maximum is at 488 nm, it's tough to deal with in a system with only this excitation wavelength. Green - 515 nm from argon, 520/530 from krypton, 532 from a doubled YAG, 543 from a green He-Ne, and 546 from a mercury lamp all work fine - excitation is necessary, at least for ready discrimination of basophils and mast cells from other cell types. However, if you are working with a basophil leukemia or mast cell line, you can look at degranulation using the orthochromatic green fluorescence excited at 488 nm. In the course of a Phase I SBIR a couple of years ago, trying to develop a simple clinical degranulation assay, I had Molecular Probes synthesize an analog of basic orange 21 with a longer wavelength absorption, which I hoped might work with a red laser; it doesn't seem to. I haven't looked into whether messing around with the rings (the dye is something close to an asymmmetric cyanine) might give shorter wavelength absorption while preserving the metachromasia. -Howard
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:02 EST