Abstract
Phase-sensitive flow cytometry (PSFC) is a technique in which fluorescence excited state decay times are measured as fluorescently labeled cells rapidly transit a finely focused, frequency-modulated laser beam. With PSFC the fluorescence lifetime is taken as a cytometric parameter to differentiate intracellular events that are challenging to distinguish with standard flow cytometry. For example PSFC can report changes in protein conformation, expression, interactions, and movement, as well as differences in intracellular microenvironments. This contribution focuses on the latter case by taking PSFC measurements of macrophage cells when inoculated with enhanced green fluorescent protein (EGFP)-expressing E. coli. During progressive internalization of EGFP-E. coli, fluorescence lifetimes were acquired and compared to control groups. It was hypothesized that fluorescence lifetimes would correlate well with phagocytosis because phagosomes become acidified and the average fluorescence lifetime of EGFP is known to be affected by pH. We confirmed that average EGFP lifetimes consistently decreased (3 to 2 ns) with inoculation time. The broad significance of this work is the demonstration of how high-throughput fluorescence lifetime measurements correlate well to changes that are not easily tracked by intensity-only cytometry, which is affected by heterogeneous protein expression, cell-to-cell differences in phagosome formation, and number of bacterium engulfed.
Highlights
Introduction ofenhanced green fluorescent protein (EGFP) into bacteria cultures was performed with an EGFP mutant DNA plasmid constructed by Dr Miho Suzuki
These data show that scattered light intensity is higher for the relatively larger RAW264.7 cells compared to the scattered light intensity of smaller E. coli and EGFP-E. coli cells indicated by events located in the top right quadrant for RAW264.7 cells and events located in the bottom left quadrant for E. coli and EGFP-E. coli cells
A variety of pH sensors[12,16,31] exist, most of the studies performed use fluorescence microscopy and the accumulation of fluorescence in cells is heterogeneous and difficult for large scale measurements such as flow cytometry. In this contribution we demonstrate how Phase-sensitive flow cytometry (PSFC) can be used to detect fluorescence lifetime-dependent changes and that those changes correlate to gradual bacteria-macrophage interactions
Summary
Introduction ofEGFP into bacteria cultures was performed with an EGFP mutant DNA plasmid constructed by Dr Miho Suzuki (generously gifted from Saitama University, Japan). We present phase-sensitive flow cytometry (PSFC) as a tool to detect phagocytosis through the measurement of the fluorescence lifetime on a cell-to-cell basis. Using a combination of fluorescence microscopy and PSFC we are able to observe the internalization of EGFP-expressing E. coli (EGFP-E. coli) by the macrophage cells and detect average fluorescence lifetime changes during this process.
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