Jun 17, 2025

Public workspaceHigh Throughput Phagocytosis assay

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gpq7qplzp/v1
  • Alexis Penverne1,2
  • 1Gandhi Lab;
  • 2University College London, University of London
Alexis Penverne
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gpq7qplzp/v1
Protocol Citation: Alexis Penverne 2025. High Throughput Phagocytosis assay. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpq7qplzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 17, 2025
Last Modified: June 17, 2025
Protocol Integer ID: 220413
Keywords: high throughput phagocytosi, longitudinal phagocytosi, phrored zymosan particle, zymosan particle, cell analysis system, incucyte, assay
Abstract
Protocol for a longitudinal phagocytosis assay using PhroRed zymosan particles (ThermoFisher ) and the Incucyte live-cell analysis system (Sartorius).
Troubleshooting
Preparing cells for assay
Differentiate microglia as described in dx.doi.org/10.17504/protocols.io.dm6gpdrp5gzp/v1
Plate microglia-like cells in a PhenoPlate 96-well (Revvity) at 150,000 cells/mL, Amount100 µL per well in X-Vivo 15 (Lonza) supplemented with 1% Glutamax, 0.5% Pen/Strep and 100ng/mL MCSF.

Wait for one week for microglia to mature
Phagocytosis assay
Incubate cells withConcentration100 ug/mL pHrodo Red Zymosan BioParticles Conjugate for Phagocytosis (ThermoFisher) in supplemented X-vivo.

Immediately place the PhenoPlate in Incucyte Live-Cell analysis system (Sartorius).
Set the analysis system to acquire images from each well of the plate at 560/585 nm and in Phase contrast. For a full plate this takes ~30 minutes, for a longitudinal assay images can be acquired every 45 minutes.
For a longitudinal assay, acquire images for 5 to 8 hours after incubation with PhrodoRed particles to observe the progress of uptake by microglia.
Note
Adding one extra acquisition step before incubating the cells with the probe can be useful. The plate can be removed in between two acquisition steps to treat with probes/drugs for longitudinal assays.

For analysis, the inbuilt pipelines of the Incucyte allow to measure the intensity, area and puncta count of the PhrodoRed signal, as well as confluence % based on Phase Contrast, making it possible to visualise the evolution of phagocytosis in cells.