Monitoring human neutrophil granule secretion by flow cytometry: secretion and membrane potential changes assessed by light scatter and a fluorescent probe of membrane potential.

Abstract

Purified human peripheral blood polymorphonuclear neutrophils (PMN) were incubated at 37 degrees 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 degrees-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 degrees-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 degrees-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, 0.998, and 0.989 for loss of 90 degrees-SC vs lysozyme, beta-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 degrees-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 degrees-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.