In response to Scott Menzie's question and in addition to Howards's suggestion of using bacteria and quenching the fluorescence of non-internalized particles... It is also possible to use fluorescently-labelled RBC to quantitate attachment and phagocytosis. The non-internalized RBC can be lysed by hypotonic shock without the need for a quenching agent which also preserves the ability to combine this assay with other phenotypic or cell surface fluorescent markers. An example of this can be found in: "Flow cytometric quantitation of attachment and phagocytosis in phenotypically-defined subpopulations of cells using PKH26-labeled FcgR-specific probes." Pricop, L. et al. Journal of Immunological Methods, vol 205, pp 55-65 (1997). regards Andy Beavis -----Original Message----- From: Howard Shapiro [mailto:hms@shapirolab.com] Sent: Thursday, January 21, 1999 7:40 PM To: cyto-inbox Subject: Re: Quenching Rhodamine Scott Menzie asks: > > Does anyone out there know a good way to quench rhodamine. Ive got a > researcher doing phagocytosis assays and needs to quench rhodamine > labeled beads which have not been phagocytized. Does trypan blue work? > If it does or anything else does what concentration do you recommend? > Thanks in advance for your help!! > For any quencher to work, the beads would have to be labeled only on the surface. The "rhodamine" beads from polysciences, and many other fluorescent beads, are impregnated with dye, which, being in the polymer matrix, is not accessible to a water-soluble quencher like trypan blue, and it would probably not be easy to find an hydrophobic quencher which would get into the beads from an aqueous solution. That's why a lot of people who do phagocytosis assays work with bacteria surface labeled by incubation with FITC, etc.; the fluorescence of these particles can be quenched. -Howard
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