Comparative study of neutrophil activation by chemiluminescence and flow cytometry.

Neutrophil activation by phorbol 12-myristate 13-acetate (PMA) and zymosan was assessed by luminol-dependent chemiluminescence (CL) in a novel microtiter plate format and by flow cytometry (FC) based on the oxidation of dihydrorhodamine 123. The results of this comparison demonstrated striking differences in kinetic parameters between these two techniques for neutrophil activation by PMA and zymosan. PMA activation, as determined by FC, was found to be an all-or-none phenomenon in that below a critical concentration of PMA, few cells were positive. Above this concentration, almost all cells were positive; however, the fluorescence intensity of positive cells increased with an increasing PMA concentration until a plateau (maximal) level was reached. In contrast, increasing zymosan concentrations resulted in proportionate increases in the percentage of positive cells until close to 100% of cells were positive. However, the fluorescence intensity of positive cells remained about the same. CL activity increased proportionately with either PMA or zymosan concentration until a maximal level was achieved. The concentration of PMA required for half-maximal activity was about 10-fold higher for FC than for CL, whereas the analogous concentration of zymosan was about 30-fold higher for CL than for FC. In addition, opsonization had only a small negative effect on the ability of zymosan to activate neutrophils, as determined by FC, whereas it had a very large enhancing effect when determined by CL. The differences in kinetic parameters of activation suggest differential sensitivity to particulate (zymosan) versus soluble (PMA) stimulants for FC and CL.

Chemiluminescent and flow cytometric analysis of gamma interferon preincubation on neonatal and adult rat polymorphonuclear leukocytes.

Gamma interferon (IFN-gamma) has multiple immunomodulating effects and has been postulated as a possible immunopotentiating agent for the prevention or treatment of neonatal infections. This report describes the effect of rat recombinant IFN-gamma on the oxidative burst activity and CD11b expression of neonatal and adult rat polymorphonuclear leukocytes (PMNL). Oxidative burst activity was assessed by chemiluminescence and dihydrorhodamine flow cytometry. Neonatal PMNL exhibited significantly less oxidative burst activity than did adult PMNL. IFN-gamma mildly enhanced the chemiluminescence response of PMNL from both the rat pups and adults, but this effect was not statistically significant when analyzed by a multivariate model of repeated-measures analysis of variance for both chemiluminescence and dihydrorhodamine flow cytometry. CD11b expression was also not significantly enhanced by IFN-gamma.