Assessment of phagosome formation and maturation by fluorescence microscopy

Phagocytosis of invading microorganisms by neutrophils is an important early response to infection. Here, we describe protocols designed for the quantitative study of particle internalization and of the subsequent maturation of the newly formed phagosomes using sheep red blood cells as a model target particle and neutrophils as phagocytes. Techniques to label the particles with fluorescent pH-sensitive dyes are presented, along with complement and immunoglobulin opsonization procedures. These labeling techniques, in combination with high-resolution digital imaging, allow for the quantitative assessment of phagocytosis at the single-cell level by bright field and fluorescence microscopy. Lastly, qualitative and quantitative methods of investigating the intraphagosomal pH are presented. We describe the use of a fluorescent weak base that accumulates in acidic cellular compartments and functions as a marker of phagosome acidification, as well as more quantitative phagosomal pH measurements by fluorescence ratio imaging. A brief description of the hardware and software components necessary for digital imaging is also provided.