A fluorescence quenching technique using trypan blue to differentiate between attached and ingested...

A fluorescence quenching method, using trypan blue, is described for quantifying the ingestion of either glutaraldehyde-fixed sheep red blood cells or antibody coated glutaraldehyde-fixed sheep red blood cells by murine peritoneal macrophages. This method is based on the observations that glutaraldehyde-fixed red blood cells fluoresce at about 585 nm when excited at 490 nm and that when trypan blue is in intimate contact with fixed red blood cells, the fluorescence is converted from a chartreuse to a red color. Thus, the ingestion of fixed erythrocytes by murine macrophages can be monitored by fluorescence microscopy after the addition of 1 mg/ml of trypan blue at the end of the assay. Extracellular glutaraldehyde-fixed red blood cells fluoresce a red color whereas the intracellular particles continue to fluoresce a chartreuse color. This method offers a simple and convenient technique for rapidly distinguishing between intracellular and extracellular glutaraldehyde-fixed red blood cells in macrophages.