Use of a microtitre plate chemiluminescence reader to study surface phagocytosis by human monocytes

Abstract

Human mononuclear cells were separated from freshly obtained peripheral venous blood by density centrifugation and the number of monocytes present estimated by volume spectroscopy. The mononuclear cells were then placed directly into the wells of a microtitre plate and incubated for one hour at 37 degrees C to promote adherence of the monocytes to the plastic wells. Non-adherent cells were then removed by washing, thus avoiding the need to treat the monocytes with EDTA or other reagents during cell preparation. The time course and dynamics of the chemiluminescence response of adherent monocytes towards opsonized zymosan was similar to those seen using non-adherent cells. The ability of adherent monocyte preparations to produce chemiluminescence following incubation for varying periods with T-lymphocyte conditioned medium was investigated. The use of a microtitre plate chemiluminescence reader allows several plates to be assayed over the 24-hour period and since small numbers of cells are required, many cultures can be analysed in one experiment. This technique (Patent applied for) promises to be a powerful tool for dissecting the cellular events which occur during macrophage activation and examining the effect of various lymphokines on the ability of monocytes to produce a chemiluminescence response.

Optimization of a phagocyte microplate chemiluminescent assay

“…6). Chemiluminescent assays are normally conducted at 37°C (5,6,14). The 78% increase in neutrophil CL peak height between 37 and 43°C was significant (t-test, p = 0.0003), as was the 56% increase in peak height for monocytes (t-test, p = 0.025).…”

A comparison of whole blood neutrophil chemiluminescence measured with cuvette and microtitre plate luminometers

“…Apart from one-sample luminometers, other instruments are now being used to measure the respiratory burst of neutrophils, such as automated, PC-controlled multiple sample luminometers and, especially recently, microplate-based luminometers are increasingly used, allowing further miniaturization and automation of the procedure (7)(8)(9)(10). Although the determination of the metabolic activity of phagocytes, either in a vial-type luminometer or in a microplate-based luminometer alone, has been described by various authors, a direct comparison of the CL activity in identical samples of phagocytes measured in parallel using both types of the luminometers has been lacking up to now.…”

Measurement of whole blood phagocyte chemiluminescence in a microtitreplate format

“…Whole blood has been used in some cases to simplify the procedure (Descamps-Latscha et al 1982;Tono-Oka et al 1983). In other studies, the recent introduction of microtitreplate luminometer has allowed for measurement of a large number of samples in a single setting (Blair et al 1988;Cree, Blair & Beck 1989). The present study was undertaken to look into the performance of zymosan-induced luminol-enhanced phagocyte chemiluminescence with the combined use of both whole blood and a microtitreplate luminometer.…”