Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Macrophage phagocytosis assay with reconstituted target particles

Nature Protocols volume 15, pages 2230–2246 (2020)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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.

References

  1. Gordon, S. & Taylor, P. R. Monocyte and macrophage heterogeneity. Nat. Rev. Immunol. 5, 953–964 (2005).

    Article  CAS  Google Scholar 

  2. Davies, L. C., Jenkins, S. J., Allen, J. E. & Taylor, P. R. Tissue-resident macrophages. Nat. Immunol. 14, 986–995 (2013).

    Article  CAS  Google Scholar 

  3. Franken, L., Schiwon, M. & Kurts, C. Macrophages: sentinels and regulators of the immune system. Cell. Microbiol. 18, 475–487 (2016).

    Article  CAS  Google Scholar 

  4. Gordon, S. Phagocytosis: an immunobiologic process. Immunity 44, 463–475 (2016).

    Article  CAS  Google Scholar 

  5. Erwig, L. P. & Gow, N. A. R. Interactions of fungal pathogens with phagocytes. Nat. Rev. Microbiol. 14, 163–176 (2016).

    Article  CAS  Google Scholar 

  6. Weiskopf, K. & Weissman, I. L. Macrophages are critical effectors of antibody therapies for cancer. mAbs 7, 303–310 (2015).

    Article  CAS  Google Scholar 

  7. Overdijk, M. B. et al. Antibody-mediated phagocytosis contributes to the anti-tumor activity of the therapeutic antibody daratumumab in lymphoma and multiple myeloma. mAbs 7, 311–320 (2015).

    Article  CAS  Google Scholar 

  8. Bain, C. C. & Mowat, A. M. Macrophages in intestinal homeostasis and inflammation. Immunol. Rev. 260, 102–117 (2014).

    Article  CAS  Google Scholar 

  9. Moore, K. J., Sheedy, F. J. & Fisher, E. A. Macrophages in atherosclerosis: a dynamic balance. Nat. Rev. Immunol. 13, 709–721 (2013).

    Article  CAS  Google Scholar 

  10. Guerreiro, R. et al. TREM2 variants in Alzheimer’s disease. N. Engl. J. Med. 368, 117–127 (2013).

    Article  CAS  Google Scholar 

  11. Hansen, D. V., Hanson, J. E. & Sheng, M. Microglia in Alzheimer’s disease. J. Cell Biol. 217, 459–472 (2018).

    Article  CAS  Google Scholar 

  12. Tay, T. L. et al. Microglia gone rogue: impacts on psychiatric disorders across the lifespan. Front. Mol. Neurosci. 10, 421 (2018).

    Article  Google Scholar 

  13. Jungi, T. W. A rapid and sensitive method allowing photometric determination of erythrophagocytosis by mononuclear phagocytes. J. Immunol. Methods 82, 141–153 (1985).

    Article  CAS  Google Scholar 

  14. Wan, C. P., Park, C. S. & Lau, B. H. S. A rapid and simple microfluorometric phagocytosis assay. J. Immunol. Methods 162, 1–7 (1993).

    Article  CAS  Google Scholar 

  15. Pacheco, P., White, D. & Sulchek, T. Effects of microparticle size and Fc density on macrophage phagocytosis. PLoS One 8, e60989 (2013).

    Article  CAS  Google Scholar 

  16. Berken, A. & Benacerraf, B. Properties of antibodies cytophilic for macrophages. J. Exp. Med 123, 119–144 (1966).

    Article  CAS  Google Scholar 

  17. Freeman, S. A. et al. Integrins form an expanding diffusional barrier that coordinates phagocytosis. Cell 164, 128–140 (2016).

    Article  CAS  Google Scholar 

  18. Getahun, A. & Cambier, J. C. Of ITIMs, ITAMs and ITAMis, revisiting Immunoglobulin Fc receptor signaling. Immunol. Rev. 268, 66–73 (2015).

    Article  CAS  Google Scholar 

  19. van der Merwe, P. A., Davis, S. J., Shaw, A. S. & Dustin, M. L. Cytoskeletal polarization and redistribution of cell-surface molecules during T cell antigen recognition. Semin. Immunol. 12, 5–21 (2000).

    Article  Google Scholar 

  20. James, J. R. & Vale, R. D. Biophysical mechanism of T-cell receptor triggering in a reconstituted system. Nature 487, 64–69 (2012).

    Article  CAS  Google Scholar 

  21. Davis, S. J. & van der Merwe, P. A. The structure and ligand interactions of CD2: implications for T-cell function. Immunol. Today 17, 177–187 (1996).

    Article  CAS  Google Scholar 

  22. Davis, S. J. & van der Merwe, P. A. The kinetic-segregation model: TCR triggering and beyond. Nat. Immunol. 7, 803–809 (2006).

    Article  CAS  Google Scholar 

  23. Bakalar, M. H. et al. Size-dependent segregation controls macrophage phagocytosis of antibody-opsonized targets. Cell 174, 131–142.e13 (2018).

    Article  CAS  Google Scholar 

  24. Chikh, G. G., Li, W. M., Schutze-Redelmeier, M.-P., Meunier, J.-C. & Bally, M. B. Attaching histidine-tagged peptides and proteins to lipid-based carriers through use of metal-ion-chelating lipids. Biochim. Biophys. Acta Biomembr. 1567, 204–212 (2002).

    Article  CAS  Google Scholar 

  25. Underhill, D. M. Macrophage recognition of zymosan particles. J. Endotoxin Res. 9, 176–180 (2003).

    Article  CAS  Google Scholar 

  26. Tavanti, A. et al. Candida albicans isolates with different genomic backgrounds display a differential response to macrophage infection. Microbes Infect. 8, 791–800 (2006).

    Article  CAS  Google Scholar 

  27. Champion, J. A. & Mitragotri, S. Role of target geometry in phagocytosis. Proc. Natl Acad. Sci. USA 103, 4930–4934 (2006).

    Article  CAS  Google Scholar 

  28. Yamauchi, A. et al. Rac2-deficient murine macrophages have selective defects in superoxide production and phagocytosis of opsonized particles. J. Immunol. 173, 5971–5979 (2004).

    Article  CAS  Google Scholar 

  29. Vidarsson, G., Dekkers, G. & Rispens, T. IgG subclasses and allotypes: from structure to effector functions. Front. Immunol. 5, 520 (2014).

    Article  Google Scholar 

  30. Jennewein, M. F. & Alter, G. The immunoregulatory roles of antibody glycosylation. Trends Immunol. 38, 358–372 (2017).

    Article  CAS  Google Scholar 

  31. Geiger, S. S., Curtis, A. M., O’Neill, L. A. J. & Siegel, R. M. Daily variation in macrophage phagocytosis is clock-independent and dispensable for cytokine production. Immunology 157, 122–136 (2019).

    Article  CAS  Google Scholar 

  32. Kamentsky, L. et al. Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software. Bioinformatics 27, 1179–1180 (2011).

    Article  CAS  Google Scholar 

  33. Edelstein, A. D. et al. Advanced methods of microscope control using μManager software. J. Biol. Methods 1, e10 (2014).

    Article  Google Scholar 

  34. Jaiswal, S., Chao, M. P., Majeti, R. & Weissman, I. L. Macrophages as mediators of tumor immunosurveillance. Trends Immunol. 31, 212–219 (2010).

    Article  CAS  Google Scholar 

  35. Willingham, S. B. et al. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc. Natl Acad. Sci. USA 109, 6662–6667 (2012).

    Article  CAS  Google Scholar 

  36. Fadok, V. A. et al. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148, 2207–2216 (1992).

    CAS  PubMed  Google Scholar 

  37. Fadok, V. A., de Cathelineau, A., Daleke, D. L., Henson, P. M. & Bratton, D. L. Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts. J. Biol. Chem. 276, 1071–1077 (2001).

    Article  CAS  Google Scholar 

  38. Segawa, K. & Nagata, S. An apoptotic ‘eat me’ signal: phosphatidylserine exposure. Trends Cell Biol. 25, 639–650 (2015).

    Article  CAS  Google Scholar 

  39. Prinz, H. Hill coefficients, dose–response curves and allosteric mechanisms. J. Chem. Biol. 3, 37–44 (2009).

    Article  Google Scholar 

  40. Hamczyk, M. R., Villa-Bellosta, R., Andrés, V., Andrés, V. & Dorado, B. In vitro macrophage phagocytosis assay. in Methods in Mouse Atherosclerosis (Springer, 2015).

  41. Koval, M., Preiter, K., Adles, C., Stahl, P. D. & Steinberg, T. H. Size of IgG-opsonized particles determines macrophage response during internalization. Exp. Cell Res. 242, 265–273 (1998).

    Article  CAS  Google Scholar 

Download references

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.

Author information

Author notes

  1. Matthew H. Bakalar

    Present address: Broad Institute of MIT and Harvard, Cambridge, MA, USA

Authors and Affiliations

  1. Department of Bioengineering and Biophysics Program, University of California Berkeley, Berkeley, CA, USA

    Aaron M. Joffe, Matthew H. Bakalar & Daniel A. Fletcher

  2. UC Berkeley/UC San Francisco Graduate Group in Bioengineering, Berkeley, CA, USA

    Aaron M. Joffe, Matthew H. Bakalar & Daniel A. Fletcher

  3. Division of Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Daniel A. Fletcher

  4. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Daniel A. Fletcher

Authors
  1. Aaron M. Joffe
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Matthew H. Bakalar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Daniel A. Fletcher
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Daniel A. Fletcher.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

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.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-020-0330-8

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing