Recording phagosome maturation through the real-time, spectrofluorometric measurement of hydrolytic activities

Abstract

The efficient degradation of internalized particulate matter is a principal objective of the macrophage's phagosome. Assessment of the true hydrolytic capacity within the phagosomal lumen is often difficult as it is subject to many factors beyond recruitment of lysosomal hydrolases. Here we outline three assays that allow quantitative measurements of serine-cysteine protease, triglyceride lipase, and beta-galactosidase activities within the phagosomes of macrophages, in real time. The assays utilize ratio fluorometry between particle-associated fluorogenic substrates and calibration fluorochromes to yield internally controlled values that record rates of substrate hydrolysis. The methods described utilize a spectrofluorometer for fluorometric measurements from a population of macrophages. These assays, however, can be expanded to high-throughput or single cell formats. In addition, this approach can be applied to measure a wide variety of phagosomal hydrolytic properties with the design of suitable fluorogenic substrates.

Fig. 1

Orientation of the BMMØ monolayer in the spectrofluorometer. Cover glass is oriented to achieve a 45° incidence with the excitation beam (excitation λ), and with the cells facing the emission slit.

Fig. 2

Phagosomal serine-cysteine protease activity in BMMØ’s. Serine-cysteine protease activity can be diminished with the serine-cysteine protease inhibitor leupeptin (100 μg/ml) and the V-ATPase inhibitor concanamycin A (100 nm).

Fig. 3

Phagosomal triglyceride lipase activity in BMMØ’s. Lipase activity can be diminished with the specific lipase inhibitor tetrahydrolipstatin (50 μg/ml) and is increased in the early phagosome by the V-ATPase inhibitor concanamycin A (100 nm).

Fig. 4

Phagosomal β-galactosidase activity in BMMØ’s. β-galactosidase activity can be diminished with the competitive inhibitor 2-phenylethyl-β-D-thiogalactoside (PETG) (200 μg/ml) and the V-ATPase inhibitor concanamycin A (100 nm).