A SINGLE ASSAY FOR MEASURING THE RATES OF PHAGOCYTOSIS AND BACTERIAL KILLING BY NEUTROPHILS

We have developed a method that enables the rates of phagocytosis and killing of bacteria by neutrophils to be measured in a single assay. N eutrophils were incubated with bacteria, and at specific intervals wer e separated from uningested bacteria by low speed centrifugation. Rate s of phagocytosis and killing were calculated from the decrease in num ber of extracellular bacteria and change in the number of intracellula r bacteria. Both phagocytosis and killing were shown to follow first-o rder kinetics, and rate constants were calculated without having to se parate the assay into two phases. In contrast to two-step methods, our method measures killing from the moment the neutrophils start ingesti ng the bacteria, and also eliminates the need to halt neutrophil activ ity temporarily and restart the assay after the extracellular bacteria have been removed. We obtained reproducible results for the phagocyto sis and killing of Staphylococcus aureus (t(1/2) = 9 min and 6 min res pectively) and Escherichia coli (t(1/2) = 10 min and 2 min respectivel y). We also were able to detect a 56% impairment in the rate of killin g of S. aureus by neutrophils from an individual with a low level of m yeloperoxidase.