Flow cytometric evaluation of nitro blue tetrazolium (NBT) reduction in human polymorphonuclear leukocytes

Abstract

Oxidative metabolic burst of activated human polymorphonuclear leukocytes (PMN) is most commonly investigated in clinical practice by evaluating nitroblue tetrazolium (NBT) reduction at the single cell level. Reduced NBT precipitates where the redox reaction has taken place and can be visualized as PMN-associated dark blue granules of formazan in light microscopy. Although widely used and not technically demanding, this method remains subjective and labor intensive, especially when large numbers of samples need to be investigated. We developed a new flow cytometry technique in which PMN membrane was rendered fluorescent by a short incubation with fluorescein-conjugated Concanavalin A. PMN were then incubated with NBT and increasing doses of a suitable stimulus, such as phorbol myristate acetate (PMA). Formazan has a distinct peak of absorption at 520 nm that represents the peak of emission of fluorescein. As a consequence, formazan quenches the PMNassociated fluorescence. Data show that a dose-dependent reduction of fluorescence can be obtained using graded amounts of PMA in normal PMN cultures. PMN-associated fluorescence remains unchanged in control patients with chronic granulomatous (CGD) disease, a disorder characterized by a selective impairment of PMN oxidative metabolism. Electronic cell size increases upon PMA incubation in normal PMN, irrespective of the presence of NBT. Conversely, forward light scatter intensity decreases in the presence, but not in the absence, of NBT indicating that the phenomenon is due to the capacity of formazan to absorb/ scatter the incident light. The present method for easily detecting NBT reducing activity at single cell level by flow cytometry makes use of commonly available, inexpensive reagents and standard instrumentation. It could become a useful test for clinical purposes.

Intranasal Delivery of an Adjuvanted Modified Live Porcine Reproductive and Respiratory Syndrome Virus Vaccine Reduces ROS Production

“…Our results are consistent with the quantitatively increased ROS production detected in the lungs of unvaccinated PRRSV-challenged pigs (32). Flow cytometric analysis was used to measure the frequency and function of ROS-producing cells (28), based on the principle that ROS mediate quenching of the cell surface fluorescent molecule, here FITC, tagged to ConA. Quenching is not directly proportional to the amount of ROS produced, mainly due to the difference in the quantity of ROS produced by each type of phagocytic cell.…”

Potentiation of human polymorphonuclear leukocyte activation by atrial natriuretic peptide. inhibitory effect of carnitine congeners

“…Thus, PMN were obtained as a total leukocyte population from peripheral blood after lysis of erythrocytes with buffered ammonium chloride. A second lysis step was performed if the cell pellet remained visibly red (20). PMN were then singled out from other blood cells using the electronic capabilities of a flow cytometer (see below).…”

Flow cytometric investigation of neutrophil oxidative burst and apoptosis in physiological and pathological situations

“…Formazan has a distinct peak of absorption at 520 nm, also the peak of fluorescein emission. As a consequence, formazan quenches the PMN-associated fluorescence (23). The major characteristics of the different nonfluorescent and fluorescent probes used to assess PMN oxidative burst using flow cytometry (fluorescence excitation and emission, specificity, sensitivity, advantages, and disadvantages) are summarized in Table 1.…”