Macrophage phagocytosis assay with reconstituted target particles

Abstract

Macrophage phagocytosis can be triggered by diverse receptor-ligand interactions to clear pathogens and dead cells from a host. Many ways of assaying phagocytosis exist that utilize a variety of phagocytic targets with different combinations of receptor-ligand interactions, making comparisons difficult. To study how phagocytosis is affected by specific changes to the target surface, we developed an in vitro assay based on reconstituted membrane-coated target particles to which known molecules can be added. The targets are made by coating glass beads with supported lipid bilayers followed by coupling proteins and other ligands of interest. Composition of the lipid bilayer can be varied to bind and orient specific proteins, incorporate signaling and reporter lipids, and control bilayer fluidity. To quantify phagocytosis, the reconstituted target particles are incubated with macrophages in vitro for a defined period of time, imaged with fluorescence microscopy and analyzed with software that measures the amount of target particle fluorescence within each macrophage. A multi-well plate format can be used for multi-parameter studies (e.g., to investigate how phagocytosis is affected by specific receptor-ligand interactions, ligand density, lipid charge, membrane fluidity and other molecular details). As an example, we demonstrate that antibody-dependent phagocytosis is more efficient for targets with fluid membranes than non-fluid membranes. The assay protocol takes approximately 6 h and requires basic molecular biology, mammalian cell culture and fluorescence microscopy skills. This assay can also be used with other phagocytic and non-phagocytic cells to study the individual or collective roles of receptors and ligands in immune effector function.

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Fig. 1: Graphical overview of the protocol.
Fig. 2: Schematic view of a reconstituted target particle with some example applications of different target configurations.
Fig. 3: Overview of steps performed during image analysis of fluorescent images.
Fig. 4: Example images and data.
Fig. 5: Demonstrations of the protocol.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Code availability

The CellProfiler project file is available in the Supplementary Software section of this paper.

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Acknowledgements

The authors thank Eva Schmid and Emily Suter for their help in refining the protocol, Sungmin Son for technical consulting and the entire Fletcher Laboratory for feedback and advice. This work was supported by the Immunotherapeutics and Vaccine Research Initiative (IVRI) at UC Berkeley and by the NSF Center for Cellular Construction. A.M.J. was funded by the Siebel Scholars Program. M.H.B. was funded by an NSF Graduate Research Fellowship and the Siebel Scholars Program. D.A.F. is a Chan-Zuckerberg Biohub investigator.

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Correspondence to Daniel A. Fletcher.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Philip van der Merwe, Suzan Rooijakkers, Eva M. Struijf and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Bakalar, M. H. et al. Cell 174, 131–142.e13 (2018): https://doi.org/10.1016/j.cell.2018.05.059

Extended data

Extended data Figure 1 Quality control checks for reconstituted targets.

a, Example images of reconstituted targets formed with DPPC-containing bilayers and POPC bilayers that have been opsonized with fluorescent antibody. Properly prepared targets should have uniform coverage of lipids and conjugated proteins. Scale bar: 5 µm. b, TIRF microscopy time courses of fluorescence recovery after photobleaching, demonstrating recovery for fluid bilayers made from POPC and no recovery for non-fluid bilayers made from DPPC. Scale bar: 25 µm. c, TIRF microscopy time courses of fluorescence recovery after photobleaching the bottom of beads coated with lipid bilayers. Fluorescence recovery is observed for fluid bilayers made from POPC, and no recovery is observed for non-fluid bilayers made from DPPC. Scale bar: 2 µm. d, Quantification of fluorescence recovery for the beads shown in c.

Extended data Figure 2 Measuring absolute density of fluorescent molecules on reconstituted target surfaces.

a, A histogram of fluorescence intensity for four samples of fluorescent beads of known MESF value for Alexa Fluor 488 obtained using flow cytometry. b, Calibration line constructed from plotting the intensity values obtained from flow cytometry and the known MESF value of the beads. The equation for the line of best fit can be used to convert flow cytometry measurements to absolute counts of Alexa Fluor 488 fluorophores, provided that all measurements are performed using the same flow cytometry settings. If the number of fluorophores per molecule of interest (i.e., protein or antibody) is known, along with the surface area of the reconstituted target, an absolute measurement of surface density for the molecule of interest can be obtained.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2.

Reporting Summary

Supplementary Software

CellProfiler project file.

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Joffe, A.M., Bakalar, M.H. & Fletcher, D.A. Macrophage phagocytosis assay with reconstituted target particles. Nat Protoc 15, 2230–2246 (2020). https://doi.org/10.1038/s41596-020-0330-8

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