Related Experiment Video
Updated: Aug 14, 2026
Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging
Published on: January 5, 2015
Fluorescent liposomes as quantitative markers of phagocytosis by alveolar macrophages
1Department of Medicine, Indiana University School of Medicine, Indianapolis 46202, USA.
Abstract:
A new phagocytic assay based on liposome ingestion by alveolar macrophages (AMs) is described. Fluorescent microspheres were encapsulated in liposomes, which allowed rapid enumeration by fluorometry. Liposomes made in the presence of vitronectin had the protein exposed on their outer surfaces, as determined by immunolabelling. Liposomes and rat AMs were incubated under conditions favorable for phagocytosis. Observation by light and electron microscopy showed AMs engulfing liposomes, with gradual transfer of fluorescent label from liposome to cell interior. This transfer was due to bona fide phagocytosis, as evidenced by (1) fluorescence of liposome-encapsulated dihydrofluorescein (DHF)-zymosan exclusively within AMs and (2) triggering of the respiratory burst by zymosan-associated liposomes only under conditions that allowed phagocytosis. Phagocytic activity was expressed as liposomes/cell, the average number of liposomes phagocytosed per macrophage. We used this technique to follow phagocytosis over time and to measure the effects of lipopolysaccharide (LPS) and vitronectin on AM phagocytosis.
Insights
A novel assay quantifies phagocytosis by alveolar macrophages (AMs) using fluorescent liposomes. This method revealed how lipopolysaccharide (LPS) and vitronectin impact AM phagocytic activity.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Alveolar macrophages (AMs) are crucial for lung immunity.
- Quantifying AM phagocytosis is essential for understanding lung defense mechanisms.
- Existing assays may not fully capture the nuances of phagocytic processes.
Purpose of the Study:
- To develop and validate a new assay for measuring liposome ingestion by AMs.
- To investigate the effects of vitronectin and lipopolysaccharide (LPS) on AM phagocytosis.
Main Methods:
- Encapsulation of fluorescent microspheres within liposomes for fluorometric quantification.
- Incubation of liposomes with rat AMs under controlled phagocytic conditions.
- Confirmation of phagocytosis via microscopy, dihydrofluorescein (DHF)-zymosan fluorescence, and respiratory burst assessment.
Main Results:
- The assay successfully enumerated liposome ingestion by AMs.
- Vitronectin exposure on liposome surfaces was confirmed via immunolabelling.
- Phagocytosis was validated by intracellular DHF-zymosan fluorescence and zymosan-induced respiratory burst.
- The assay allowed measurement of phagocytic activity (liposomes/cell) and the impact of LPS and vitronectin.
Conclusions:
- A robust and quantifiable assay for AM phagocytosis using fluorescent liposomes has been established.
- The assay provides insights into the mechanisms of phagocytosis and the influence of specific molecules like vitronectin and LPS.
