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Updated: Aug 1, 2026
High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization
Published on: November 27, 2014
Flow cytometric, phase-resolved fluorescence measurement of propidium iodide uptake in macrophages containing
J A Steinkamp1, Y E Valdez, B E Lehnert
1Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico. steinkamp@lanl.gov
Background:
Spectral interference (overlap) from phagocytosed green-yellow (GY) microspheres in the flow cytometric, red fluorescence emission measurement channel causes errors in quantifying damaged/dead alveolar macrophages by uptake of propidium iodide.
Methods:
Particle burdens of uniform GY fluorescent microspheres phagocytosed by rat alveolar macrophages and the discrimination of damaged/dead cells as indexed by propidium iodide uptake were assessed with conventional and phase-sensitive flow cytometry.
Results:
The fluorescence spectral emission from phagocytosed microspheres partly overlapped the propidium iodide red fluorescence emission and interfered with the measurement of damaged/dead cells when using conventional flow cytometry without subtractive compensation. This caused errors when estimating the percentage of nonviable, propidium iodide-positive, phagocytic macrophages. The interference was eliminated by employing phase-sensitive detection in the red fluorescence measurement channel based on differences in fluorescence lifetimes between the fluorescent microspheres and propidium iodide. Intrinsic cellular autofluorescence, whose fluorescence lifetime is approximately the same as that of the phagocytosed microspheres, also was eliminated in the phase-sensitive detection process. Because there was no detectable spectral interference of propidium iodide in the green fluorescence (phagocytosis) measurement channel, conventional fluorescence detection was employed.
Conclusions:
Phase-resolved, red fluorescence emission measurement eliminates spectral overlap errors caused by autofluorescent phagocytes that contain fluorescent microspheres in the analyses of propidium iodide uptake. Cytometry 39:45-55, 2000. Published 2000 Wiley-Liss, Inc.
Insights
Phase-sensitive flow cytometry eliminates spectral overlap errors when quantifying damaged alveolar macrophages using propidium iodide. This method accurately measures cell viability by distinguishing fluorescent microspheres from cellular signals.
Area of Science:
- Cellular biology
- Flow cytometry techniques
- Fluorescence spectroscopy
Background:
- Flow cytometry is crucial for analyzing cell populations.
- Spectral overlap from phagocytosed microspheres can interfere with cell viability assays.
- Accurate quantification of damaged alveolar macrophages is essential in research.
Purpose of the Study:
- To assess the impact of spectral interference on cell viability measurements.
- To evaluate the effectiveness of phase-sensitive flow cytometry in overcoming these interferences.
- To improve the accuracy of quantifying damaged alveolar macrophages.
Main Methods:
- Utilized conventional and phase-sensitive flow cytometry.
- Assessed particle burdens of green-yellow (GY) fluorescent microspheres in rat alveolar macrophages.
- Measured propidium iodide uptake as an indicator of cell damage/death.
Main Results:
- Conventional flow cytometry without compensation showed spectral overlap errors.
- Phase-sensitive detection eliminated interference from microspheres and autofluorescence.
- Accurate measurement of propidium iodide uptake was achieved using phase-sensitive methods.
Conclusions:
- Phase-resolved fluorescence measurement effectively removes spectral overlap errors.
- This technique enhances the accuracy of analyzing propidium iodide uptake in phagocytic cells.
- Phase-sensitive flow cytometry provides a reliable method for cell viability studies.
