EVALUATION OF A FLOW CYTOMETRIC FLUORESCENCE QUENCHING ASSAY OF PHAGOCYTOSIS OF SENSITIZED SHEEP ERYTHROCYTES BY POLYMORPHONUCLEAR LEUKOCYTES

A number of reports have been published describing phagocytosis assays for flow cytometric analysis. In some of these, the fluorescence quen ching technique has been used to discriminate between adherent and ing ested particles. In this report, we have evaluated the efficacy of a q uantitative fluorescence quenching technique with crystal violet and t rypan blue for application in a phagocytosis assay with polymorphonucl ear leukocytes and sensitized sheep red blood cells. We set the requir ements to a high quenching efficiency of the fluorescence of extracell ularly bound particles and no intracellular quenching. The latter was determined using polymorphonuclear leukocytes stained with the fluores cent nuclear dye hydroethidine. We observed that both trypan blue and crystal violet efficiently quench the fluorescence of PKH26 (a red flu orescent membrane-associated dye) erythrocytes but that only crystal v iolet quenches intracellular fluorescence. In testing trypan blue and crystal violet from different manufacturers, there was no real differe nce between different brands of crystal violet, but only the trypan bl ue from Merck turned out to be an efficient quencher, whereas the othe r brands of trypan blue showed low quenching efficiency, Trypan blue a t a concentration of 25-50 mu g/ml proved to be a good quencher of the fluorescent erythrocytes and exerted minimal side effects: over 90% q uenching of the erythrocyte, no intracellular quenching, moderate incr ease in autofluorescence of the polymorphonuclear leukocytes, and no c ell loss. (C) 1994 Wiley-Liss, Inc.