Phagocytic and oxidative burst activities of neutrophils and monocytes

In order to determine the phagocytosis of neutrophils and monocytes, Phagotest^® kit was used. The kit contains fluorescein-labeled opsonized E. coli bacteria and other reagents, as reported in the manufacturer’s description. Briefly, heparinized whole blood, processed within 2 h of sampling, was incubated with the FITC-labeled E. coli bacteria at 37°C and a negative control sample remained on ice. The phagocytosis was stopped by placing the samples on ice and adding quenching solution. This solution allowed the discrimination between attachment and internalization of bacteria by quenching the FITC fluorescence of surface bound bacteria leaving the fluorescence of internalized particles unaltered. After two washing steps with washing solution erythrocytes were then removed by addition of lysing solution. Data were analyzed by flow cytometry (BD FACS Canto^™) evaluating 2 parameters as recommended in the kit: 1) the proportion of cells undergoing phagocytosis in general (phagocytosis rate, P-R), expressed as percentage and indicating the ingestion of one or more bacteria per cell, and 2) the individual phagocytic activity per cell (the number of bacteria engulfed per cell), corresponding to the median fluorescence intensity (P-MFI). The number of cells recorded per experiment ranged between 15.000 and 20.000. An example of the gating strategy is shown in S1 Fig.

In order to measure the oxidative burst activity of neutrophils and monocytes, Phagoburst kit^® was employed. The kit contains unlabeled opsonized E. coli bacteria as particulate stimulus, the protein kinase C ligand Phorbol 12-Myristate 13-Acetate (PMA) as high stimulus, the chemotactic peptide N-formyl-MetLeuPhe (fMLP) as low physiological stimulus, dihydrorhodamine (DHR)-123 as a fluorogenic substrate and other reagents as reported in the manufacturer’s description. Briefly, heparinized whole blood, processed within 2 h of sampling, was incubated with the various stimuli at 37°C, a sample without stimulus served as negative control. Upon stimulation with unlabeled E.coli, neutrophils and monocytes produced reactive oxygen metabolites (superoxide anion, hydrogen peroxide, hypochlorous acid) which destroyed bacteria inside the phagosome. Formation of the reactive oxidants during the oxidative burst has been monitored by the addition and oxidation of DHR-123. The reaction was stopped by addition of lysing solution, which removed erythrocytes and allowed a partial fixation of cells. Data were analyzed by flow cytometry (BD FACS Canto^™) measuring 2 parameters: 1) the proportion of cells having produced reactive oxygen radicals (burst rate, B-R), expressed as percentage, and 2) the burst enzymatic activity per cell, corresponding to the median fluorescence intensity (B-MFI). The number of cells recorded per experiment ranged between 15.000 and 20.000.