Neutrophils, which constitute 50 – 70% of circulating white blood cells, are much more numerous than other granulocytes namely eosinophils (1% - 3%) and basophils (<1 %) and are the first cells recruited from the bloodstream to sites of infection.

Following activation by bacterial byproducts or other immune stimuli - such as LPS, fMLP, PMA - neutrophils execute several specialized functions that include chemotaxis, phagocytosis, and the generation of reactive oxygen species (ROS). PMA, an analog of DAG, stimulates ROS production by activation of protein kinase C, whereas fMLP is a G-protein coupled receptor agonist.

The primary aim of this study was to investigate the use of DHR in a flow cytometric assay as a semi-quantitative measure of the respiratory burst in neutrophils.

Heparinized blood was obtained from healthy adult volunteers. Neutrophils were isolated by centrifugation on Percoll gradient followed by hypotonic lysis of residual red blood cells. Five aliquots of 4x10⁵ cells each were activated for 30 minutes with either fMLP at 10 nM, 100 nM or 1000 nM, PMA at 100nM, or no activation (control). Prior to activation, the aliquots were incubated with dihydrorhodamine 123 for 5 minutes at room temperature.

The samples were then analyzed using a flow cytometer (FACScalibur) and CellQuest software. The forward and side light scatter profiles were adjusted to ensure that the neutrophil population was clearly displayed. Fluorescence was measured on the FL1 green channel (wavelength 530 nm). A total of 10,000 events were collected for each sample.

One of the greatest advantages of this assay over other conventional assays – such as spectrophotometry, luminol-based assays - is the ability to measure oxidative burst at the single cell level. Therefore, the percentage of cells presenting fluorescence as a measure of respiratory burst higher than control cells may be determined. In this regard, our results have shown that activation with fMLP resulted in an increased number of ROS-producing cells by 51.9%, whereas PMA activated almost every cell in the sample (99.7%).

Furthermore, increasing concentrations of fMLP gave significantly higher number of stimulated cells (from 31.53% to 61.50%), although not higher fluorescence. Kinetic studies also show that the time required for stimulation with fMLP is significantly larger than with PMA.