2.2.1. Neutrophil Respiratory Burst Assay (BURSTTEST)

Flow cytometry was used to evaluate the neutrophil respiratory burst with a BURSTTEST reagent kit (ORPEGEN Pharma, Heidelberg, Germany). A FACSCalibur cytometer with argon laser at a wavelength of 488 nm (Becton Dickinson, CA, USA) was applied in order to examine the ROS production. The CELLQuest programme (Becton Dickinson) was used to analyze the achieved results that were shown as the median fluorescence intensity (MFI) of phorbol 12-myristate 13-acetate (PMA) or E. coli-stimulated neutrophils.

A quantitative determination of the neutrophil oxidative burst was possible due to the BURSTTEST; dihydrorhodamine (DHR) 123 served as a fluorogenic substrate while unlabeled opsonized E. coli bacteria were used as the particulate stimulus. PMA or E. coli was incubated with heparinized whole blood at 37 °C and as a negative background control a sample without stimulus was used. ROS (i.a., hydrogen peroxide, hypochlorous acid, and superoxide anion) were produced by neutrophils upon stimulation and they were responsible for intraphagosomal bacterial killing; this phenomenon takes place in the mitochondria as DHR 123 can be oxidized by H₂O₂ and O₂⁻ to rhodamine 123, and then monitored following the reactive oxidants synthesis during respiratory burst in the neutrophils. Next, a bright fluorescent signal upon excitation by blue light (488 nm) was emitted as a consequence of the biochemical reaction described above. It was stopped by the addition of LYSING SOLUTION, which removes erythrocytes and contributes to a partial neutrophil fixation. DNA STAINING SOLUTION is added to exclude aggregation artifacts of bacteria or cells after one washing step with WASHING SOLUTION. The MFI following the interaction of reactive oxygen radicals is then measured.

The BURSTTEST assay procedure has been thoroughly described by the authors of this paper in their earlier report [19].