Objective

Phagocytosis plays an essential role in host defence and the maintenance of tissue homeostasis. Particles labelled with pH-sensitive fluorescent dyes have great utility for monitoring phagocytic events and distinguishing these from particle binding. However, as a significant part of the fluorescent signal for these probes is generated by the acidification of the endocytic vacuole, rather than the particle internalisation event itself, with some cargos, drug treatments or as a result of host cell heterogeneity, vacuoles may not acidify, or acidify at different rates. To perform quantitative measures of phagocytosis under these conditions, we have used confocal microscopy to obtain Z-stacks of the internalising cell and particle. Analysis of these slices by maximum projections provides a simple way to quantify internalisation with a relatively light computational workload, and we present here the results of a small library screen that identified modulators of particle internalisation. However, to obtain a ‘truer’ measure of internalisation, we also present the 3-dimensional analysis of the internalising cell volume. This allows the measurement of the internalised particle within the internalising cell without relying on pH-dependent change in fluorescence, while still giving good discrimination from particle binding events that occur at the cell surface. This can be performed in 96- or 384-well plates to allow the testing of compounds that may modify phagocytosis or internalisation events that are not connected to major changes in the pH of the endocytic vacuole.