Monitoring Human Neutrophil Granule Secretion by Flow Cytometry: Secretion and Membrane Potential Changes Assessed by Light Scatter and a Fluorescent Probe of Membrane Potential

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Abstract
Purified human peripheral blood polymorphonuclear neutrophils (PMN) were incubated at 37°C with the fluorescent membrane potential sensitive cyanine dye di-O-C(5)(3) and exposed to a number of stimulatory agents (N- formylmethionylleucylphenylalanine (FMLP), cytochalasin B (cyto B) + FMLP, phorbol myristate acetate (PMA). Flow cytometry was utilized to measure changes in forward light scatter (FS), orthogonal light scatter (90°-SC), and fluorescence intensity of individual cells over time. A saturating (10 − 6 M) dose of FMLP lead to a significant increase in the cells' FS without a change in 90°- SC as well as a heterogeneous loss of di-O-C(5)(3) fluorescence. PMA (100 ng/ ml) also caused an increase in FS but a uniform loss of dye fluorescence by all cells (apparent depolarization). Cyto B + FMLP produced an increase in FS, a marked loss of 90°-SC, and a uniform loss of fluorescence. Secretion experiments under identical incubation conditions indicated a significantly positive relationship between loss of enzyme markers or cell granularity and orthogonal light scatter (r = 0.959, 0.998f and 0.989 for loss of 90°-SC vs lysozyme, 0- glucuronidase, and granularity index, respectively). Sequential exposure of PMN to PMA and then cyto B + FMLP produced a stepwise shift in scatter parameters (increased FS then loss of 90°-SC). Normalization of membrane potential dye fluorescence changes for the changes in light scatter did not abrogate the heterogeneous fluorescence response of cells to stimulus, indicating that stimulus-induced scatter changes were not responsible for such fluorescence shifts. The data demonstrate that loss of 90°-SC relates closely to secretion of primary granules while changes in FS reflect alterations in cell shape and/or surface/volume ratios that accompany cell activation. Flow cytometric light scatter measurements may yield important information on the extent of prior cell degranulation or activation.