Immunofluorescence and Confocal Microscopy of Neutrophils

Neutrophils are short-lived granulocytes essential for innate host defense. We describe here methods for analysis of resting and activated cells using immunofluorescence and confocal microscopy. Procedures for stimulation of adherent and suspended cells are provided along with protocols for particle opsonization and synchronized phagocytosis. Most importantly, we describe in detail methods for rapid and efficient cell fixation and permeabilization that optimize detection of granule proteins and NADPH oxidase components. Variables that impact antigen detection (such as cell spreading, degranulation, and phagocytosis) are discussed as are methods for image acquisition and analysis.