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Updated: Jun 7, 2026
High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization
Published on: November 27, 2014
Quantification of microsized fluorescent particles phagocytosis to a better knowledge of toxicity mechanisms
L Leclerc1, D Boudard, J Pourchez
1LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées, F-42023, Saint-Etienne, France. leclerc@emse.fr
Background:
The use of micro- or nanometric particles is in full expansion for the development of new technologies. These particles may exhibit variable toxicity levels depending on their physicochemical characteristics. We focused our attention on macrophages (MA), the main target cells of the respiratory system responsible for the phagocytosis of the particles. The quantification of the amount of phagocytosed particles seems to be a major element for a better knowledge of toxicity mechanisms. The aim of this study was to develop a quantitative evaluation of uptake using both flow cytometry (FCM) and confocal microscopy to distinguish entirely engulfed fluorescent microsized particles from those just adherent to the cell membrane and to compare these data to in vitro toxicity assessments.
Methods:
Fluorescent particles of variable and well-characterised sizes and surface coatings were incubated with MA (RAW 264.7 cell line). Analyses were performed using confocal microscopy and FCM. The biological toxicity of the particles was evaluated [lactate dehydrogenase (LDH) release, tumor necrosis factor (TNF)-α, and reactive oxygen species (ROS) production].
Results And Conclusion:
Confocal imaging allowed visualization of entirely engulfed beads. The amount of phagocytic cells was greater for carboxylate 2-µm beads (49 ± 11%) than for amine 1-µm beads (18 ± 5%). Similarly, side scatter geometric means, reflecting cellular complexity, were 446 ± 7 and 139 ± 12, respectively. These results confirm that the phagocytosis level highly depends on the size and surface chemical groups of the particles. Only TNF-α and global ROS production varied significantly after 24-h incubation. There was no effect on LDH and H(2)O(2) production.
Insights
Particle uptake by macrophages depends on size and surface chemistry. This study quantifies phagocytosis using flow cytometry and microscopy, revealing key factors influencing particle interaction and toxicity.
Area of Science:
- Nanotechnology
- Materials Science
- Cell Biology
Background:
- Micro- and nanometric particles are increasingly used in new technologies.
- Particle toxicity varies with physicochemical properties.
- Macrophages (MA) are key respiratory cells for particle phagocytosis.
Purpose of the Study:
- Quantify particle uptake by MA to understand toxicity.
- Develop methods to distinguish engulfed particles from surface-adherent ones.
- Correlate particle uptake with in vitro toxicity.
Main Methods:
- Incubation of MA (RAW 264.7) with fluorescent particles of varying sizes and coatings.
- Analysis using confocal microscopy and flow cytometry (FCM).
- Assessment of biological toxicity: LDH release, TNF-α, and ROS production.
Main Results:
- Confocal imaging visualized entirely engulfed particles.
- Phagocytosis was higher for 2-µm carboxylate beads (49%) than 1-µm amine beads (18%).
- Side scatter in FCM indicated greater cellular complexity with carboxylate beads.
Conclusions:
- Phagocytosis efficiency is significantly influenced by particle size and surface chemistry.
- TNF-α and ROS production varied significantly after 24-h incubation.
- No significant effects on LDH or H(2)O(2) production were observed.
