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High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization
Published on: November 27, 2014
Development of novel fluorescent particles applicable for phagocytosis assays with human macrophages
Cecilia Sóñora1,2, Paula Arbildi1, Iris Miraballes-Martínez3
1a Cátedra de Inmunología, Instituto de Química Biológica - Facultad de Ciencias y Departamento de Biociencias - Facultad de Química, Universidad de la República , Montevideo, Uruguay.
Abstract:
Phagocytosis is a fundamental process for removal of pathogens and for clearance of apoptotic cells. The objective of this work was the preparation of fluorescent microspheres by a simple method and the evaluation of its applicability in phagocytosis assays by using different human derived cells, differentiated THP-1 cell line and blood monocytes, with flow cytometry measurements for functionality assays. Our results show that microparticles are efficiently internalised in a non-opsonised form and in dose-dependent manner by both cellular types. Concerning mechanism we determined that tTG-β3 integrin signaling could be involved in the uptake of these particles.
Insights
Researchers developed simple fluorescent microspheres for phagocytosis assays. These particles are efficiently internalized by human immune cells, suggesting potential for studying cellular uptake mechanisms involving tTG-β3 integrin signaling.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Phagocytosis is a critical cellular process for pathogen elimination and apoptotic cell clearance.
- Developing reliable tools for studying phagocytosis is essential for understanding immune responses and developing therapies.
Purpose of the Study:
- To prepare simple fluorescent microspheres for phagocytosis assays.
- To evaluate the applicability of these microspheres using human-derived cells, including THP-1 cells and blood monocytes.
- To investigate the underlying mechanisms of particle uptake.
Main Methods:
- Simple preparation of fluorescent microspheres.
- Phagocytosis assays using differentiated THP-1 cells and primary human blood monocytes.
- Flow cytometry for quantifying particle internalization and assessing cellular functionality.
Main Results:
- Fluorescent microspheres were efficiently internalized by both THP-1 cells and monocytes in a dose-dependent manner, even without opsonization.
- Flow cytometry confirmed high uptake rates, indicating the microspheres' suitability for phagocytosis assays.
- Evidence suggests that tissue transglutaminase beta-3 (tTG-β3) integrin signaling may play a role in the uptake mechanism.
Conclusions:
- The developed fluorescent microspheres are effective tools for evaluating phagocytosis in human immune cells.
- The findings highlight the potential involvement of tTG-β3 integrin signaling in the non-opsonic uptake of particles.
- This method provides a valuable approach for functional phagocytosis assays and mechanistic studies.
