10 May 2001 Flow cytometric phase-resolved discrimination of damaged/dead cells by propidium iodide uptake in macrophages having phagocytized fluorescent microspheres
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Proceedings Volume 4252, Advances in Fluorescence Sensing Technology V; (2001) https://doi.org/10.1117/12.426730
Event: BiOS 2001 The International Symposium on Biomedical Optics, 2001, San Jose, CA, United States
Abstract
Instilled particle burdens of uniform green-yellow fluorescent microspheres phagocytized by rat alveolar (lung) macrophages and cell viability, as indexed by propidium iodide uptake (red fluorescence), were assessed using flow cytometry. Since the spectral emission from phagocytized microspheres partially overlapped the propidium iodide fluorescence and interfered with the conventional flow cytometric measurement of damaged/dead cells without subtractive compensation, this caused errors when estimating the percentage of non-viable, propidium iodide positive, phagocytic macrophages. The interference was eliminated by employing phase-sensitive detection in the red fluorescence measurement channel based on differences in lifetimes between the fluorescent microspheres and propidium iodide. In addition, intrinsic cellular autofluorescence, whose fluorescence lifetime is approximately the same as the phagocytized microspheres, also was eliminated in the measurement process. Since there was no detectable spectral interference of propidium iodide in the green fluorescence (particle phagocytosis) measurement channel, conventional fluorescence detection was employed.
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John A. Steinkamp, Yolanda E. Valdez, Bruce E. Lehnert, "Flow cytometric phase-resolved discrimination of damaged/dead cells by propidium iodide uptake in macrophages having phagocytized fluorescent microspheres", Proc. SPIE 4252, Advances in Fluorescence Sensing Technology V, (10 May 2001); doi: 10.1117/12.426730; https://doi.org/10.1117/12.426730
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