Evaluation of a flow cytometric fluorescence quenching assay of phagocytosis of sensitized sheep erythrocytes by polymorphonuclear leukocytes

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Abstract
A number of reports have been published describing phagocytosis assays for flow cytometric analysis. In some of these, the fluorescence quenching technique has been used to discriminate between adherent and ingested particles. In this report, we have evaluated the efficacy of a quantitative fluorescence quenching technique with crystal violet and trypan blue for application in a phagocytosis assay with polymorphonuclear leukocytes and sensitized sheep red blood cells. We set the requirements to a high quenching efficiency of the fluorescence of extracellularly bound particles and no intracellular quenching. The later was determined using polymorphonuclear leukocytes stained with the fluorescent nuclear dye hydroethidine. We observed that both trypan blue and crystal violet efficiently quench the fluorescence of PKH26 (a red fluorescent membrane-associated dye) erythrocytes but that only crystal violet quenches intracellular fluorescence. In testing trypan blue and crystal violet from different manufacturers, there was no real difference between different brands of crystal violet, but only the trypan blue from Merck turned out to be an efficient quencher, whereas the other brands of trypan blue showed low quenching efficiency. Trypan blue at a concentration of 25–50 μg/ml proved to be a good quencher of the fluorescent erythrocytes and exerted minimal side effects: over 90% quenching of the erythrocytes, no intracellular quenching, moderate increase in autofluorescence of the polymorphonuclear leukocytes, and no cell loss.