ImageStream cytometry extends the analysis of phagocytosis and oxidative burst

Phagocytosis is often measured using conventional microscopy and flow cytometry. ImageStream cytometry is a new technology that combines the advantages of both methods, enabling statistically robust microscopic applications. We compared ImageStream cytometry to flow cytometry in a whole blood model of phagocytosis with viable, fluorescence-marked Staphylococcus aureus. We furthermore measured the co-localization of intracellular bacteria to sites of oxidative burst, as well as changes in cell size and actin levels as a result of phagocytosis.

Fluorescence-labeled S. aureus in a ratio of 5:1 bacteria per leukocyte were added to whole blood. Phagocytosis was stopped at different time points. After staining of neutrophils and lysis of erythrocytes, samples were analysed by ImageStream cytometry and flow cytometry.

Phagocytosis and oxidative burst determined by flow cytometry and ImageStream cytometry showed strong correlation. In contrast to flow cytometry, ImageStream cytometry easily detected and excluded extracellular adherent bacteria from the measurement of phagocytosis, and enumerated the bacteria within each neutrophil. Using the Bright Detail Similarity score, we identified a subset of neutrophils with intracellular bacteria co-localized to sites of oxidative burst activity. Phagocytosis resulted in an increase in cell size and actin polymerization as determined by an increase in phalloidin fluorescence intensity.

We describe a simple whole blood image-based method for measuring bacterial phagocytosis and oxidative burst. ImageStream cytometry provides the spatial resolution to determine the number of bacteria ingested and the sub-cellular localization and trafficking patterns that enables a more complete evaluation of the phagocytic process.