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Updated: May 13, 2026
An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons
Published on: June 8, 2014
Chloride channel blockers suppress formation of engulfment pseudopodia in microglial cells
Barbara Harl1, Judith Schmölzer, Martin Jakab
1Department of Cell Biology, University of Salzburg, Salzburg, Austria.
Background/Aims:
Phagocytosis depends on the formation of engulfment pseudopodia surrounding the target. We tested in microglia, monocyte-derived cells in the brain, whether a swelling-activated Cl(-)-current (I(Cl,swell)), required for global cell volume (CV) regulation, also contributes to local expansion and retraction of engulfment pseudopodia.
Methods:
We used scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) to visualize and quantify the uptake of polystyrene microbeads (MBs) by microglial cells. Flow cytometry was used for cell volume measurments and I(Cl,swell) was measured by whole-cell patch clamp.
Results:
We found that exposure of microglial BV-2 cells to MBs in Cl(-)-free extracellular solution attenuated MB uptake and that the Cl(-)-channel blockers DIOA, flufenamic acid, NPPB and DCPIB suppressed the uptake of MBs in BV-2 cells and in primary microglial cells. Microglial cells exposed to MBs in the presence of Cl(-) channel blockers failed to extend engulfment pseudopodia. We observed that cells containing at least three MBs revealed an about twofold increase in current density of I(Cl, swell) compared to cells without MB. Osmotic challenges to stimulate global CV regulation before exposure to MBs modulated phagocytosis. Pre-conditioning of cells in hypo- or hypertonic medium for 12-16 hours caused a decrease in MB uptake.
Conclusion:
These findings indicate that I(Cl,swell) contributes to formation of engulfment pseudopodia and participates in engulfment and particle uptake in microglial cells.
Insights
A swelling-activated chloride current (I(Cl,swell)) is crucial for microglial cell engulfment. This current supports the formation of pseudopodia, enabling phagocytosis of particles by these brain immune cells.
Area of Science:
- Cell Biology
- Neuroscience
- Immunology
Background:
- Phagocytosis, a key immune process, relies on the formation of engulfment pseudopodia.
- Microglia, the brain's resident immune cells, perform phagocytosis.
- Swelling-activated chloride currents (I(Cl,swell)) regulate cell volume.
Purpose of the Study:
- To investigate the role of swelling-activated chloride currents (I(Cl,swell)) in microglial phagocytosis.
- To determine if I(Cl,swell) influences the local expansion and retraction of engulfment pseudopodia during phagocytosis.
Main Methods:
- Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) for visualizing microbead uptake.
- Flow cytometry for cell volume measurements.
- Whole-cell patch clamp electrophysiology to measure I(Cl,swell).
Main Results:
- Inhibition of chloride currents or chloride-free solutions reduced microbead uptake by microglial cells.
- Chloride channel blockers prevented the extension of engulfment pseudopodia.
- Increased I(Cl,swell) current density was observed in cells actively engulfing microbeads.
Conclusions:
- Swelling-activated chloride currents (I(Cl,swell)) are essential for engulfment pseudopodia formation in microglia.
- I(Cl,swell) plays a significant role in the phagocytic process and particle uptake by microglial cells.
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