Dataset Information

Real-Time Monitoring of Lysosomal Membrane Permeabilization Using Acridine Orange.

ABSTRACT: Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of lysosomes often precede apoptotic or necrotic cell death and occur during both physiological and pathological conditions. The weak base acridine orange readily enters cells and accumulates in the acidic environment of lysosomes. Vital staining with acridine orange is a well-proven technique to observe lysosomal destabilization using fluorescence microscopy and flow cytometry. These analyses are, however, time consuming and only adapted for discrete time points, which make them unsuitable for large-scale approaches. Therefore, we have developed a time-saving, high-throughput microplate reader-based method to follow destabilization of the lysosomal membrane in real-time using acridine orange. This protocol can easily be adopted for patient samples since the number of cells per sample is low and the time for analysis is short.

SUBMITTER: Eriksson I

PROVIDER: S-EPMC10459729 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Real-Time Monitoring of Lysosomal Membrane Permeabilization Using Acridine Orange.

Eriksson Ida I Vainikka Linda L Persson Hans Lennart HL Öllinger Karin K

Methods and protocols 20230809 4

Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of lysosomes often precede apoptotic or necrotic cell death and occur during both physiological and pathological conditions. The weak base acridine orange readily enters cells and accumulates in the acidic environment of lysosomes. Vital staining with acridine orange is a well-proven technique to observe lysosomal destabilization usi ...[more]

PMID: 37623923

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Project description:Mucus secretion is the first-line of defence against the barrage of irritants inhaled into human lungs, but abnormally thick and viscous mucus results in many respiratory diseases. Understanding the processes underlying mucus pathology is hampered, in part, by lack of appropriate experimental tools for labeling and studying mucin granule secretion from live cells with high sensitivity and temporal resolution. In this report we present original spectroscopic properties of acridine orange (AO) which could be utilized to study granule release and mucin swelling with various advanced fluorescence imaging approaches. Low concentration (<200 μM) AO solutions presented absorption maximum at 494 nm, emission maximum at 525 nm and only ∼1.76 ns fluorescence lifetime. By contrast at high concentrations (4-30 mM) favoring formation of AO aggregates, a very different absorption with maximum at ∼440 nm, dramatically red-shifted emission with maximum at 630 nm, and over 10-fold increased fluorescence lifetime (∼20 ns) was observed. To verify potential utility of AO for real-time imaging we have performed confocal, total internal reflection fluorescence (TIRF) and fluorescence lifetime imaging (FLIM) of AO-stained Calu-3 cells. We found similar red-shifted fluorescence spectra and long fluorescence lifetime in intracellular granules as compared to that in the cytoplasm consistent with granular AO accumulation. Mechanical stimulation of Calu-3 cells resulted in multiple exocytotic secretory events of AO-stained granules followed by post-exocytotic swelling of their fluorescently-labeled content that was seen in single-line TIRF images as rapidly-expanding bright-fluorescence patches. The rate of their size expansion followed first-order kinetics with diffusivity of 3.98±0.07×10(-7)c m(2)/s, as expected for mucus gel swelling. This was followed by fluorescence decrease due to diffusional loss of AO that was ∼10-fold slower in the secreted mucus compared to bulk aqueous solution. In summary, we showed that AO-staining could be utilized for real-time TIRF imaging of mucin granule exocytosis and mucin swelling with high sensitivity and temporal resolution. Considering unique AO fluorescence properties that permit selective excitation of AO monomers versus aggregates, our study lays the groundwork for future development of two-color excitation scheme and two-color fluorescence FLIM live-cell imaging assay with potentially many biological applications.

Dataset Information

Real-Time Monitoring of Lysosomal Membrane Permeabilization Using Acridine Orange.

Publications

Real-Time Monitoring of Lysosomal Membrane Permeabilization Using Acridine Orange.

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