Introduction to Fluorescence Probing of Biological Membranes

Demchenko, Alexander P.; Duportail, Guy; Oncul, Sule; Klymchenko, Andrey S.

doi:10.1007/978-1-4939-1752-5_3

Cited by 23 publications

(11 citation statements)

References 112 publications

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“…Using these criteria, we compared the performance of four commonly used dyes: Di-8-ANEPPS, FM1-43, Dioc14(3), and Membright-488, a recently developed dye. Whereas Di-8-ANEPPS and FM1-43 contain pyridinium-based scaffolds (see Figure S1) that promote complete intercalation into a vesicle lipid bilayer, Dioc14(3) and Membright-488 are dialkylcarbocyanine derivatives with long hydrocarbon side chains that anchor the dye into a vesicle’s surface (see Figure S2).…”

Section: Resultsmentioning

confidence: 99%

Sizing Extracellular Vesicles Using Membrane Dyes and a Single Molecule-Sensitive Flow Analyzer

et al. 2021

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Extracellular vesicles (EVs) are membranous particles released by most cells in our body, which are involved in many cell-to-cell signaling processes. Given the nanometer sizes and heterogeneity of EVs, highly sensitive methods with single-molecule resolution are fundamental to investigating their biophysical properties. Here, we demonstrate the sizing of EVs using a fluorescence-based flow analyzer with single-molecule sensitivity. Using a dye that selectively partitions into the vesicle’s membrane, we show that the fluorescence intensity of a vesicle is proportional to its diameter. We discuss the constraints in sample preparation which are inherent to sizing nanoscale vesicles with a fluorescent membrane dye and propose several guidelines to improve data consistency. After optimizing staining conditions, we were able to measure the size of vesicles in the range ∼35–300 nm, covering the spectrum of EV sizes. Lastly, we developed a method to correct the signal intensity from each vesicle based on its traveling speed inside the microfluidic channel, by operating at a high sampling rate (10 kHz) and measuring the time required for the particle to cross the laser beam. Using this correction, we obtained a threefold greater accuracy in EV sizing, with a precision of ±15–25%.

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Section: Resultsmentioning

confidence: 99%

Sizing Extracellular Vesicles Using Membrane Dyes and a Single Molecule-Sensitive Flow Analyzer

et al. 2021

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“…Fluorescence probing is one of the most powerful and commonly used tools in the studies of biomembrane structure and dynamics that can be applied on different levels, from lipid monolayers and bilayers to living cells, tissues and whole bodies (Demchenko et al 2009 ). Being smaller than the membrane width, functional organic dyes can characterize the microscopic analogs of viscosity, polarity, and hydration, as well as the molecular order, environment relaxation, and electrostatic potentials at the sites of their location (Demchenko et al 2015 ).…”

Section: Sensing Within the Cell Membranementioning

confidence: 99%

Sensing Inside the Living Cells

Demchenko

2015

Introduction to Fluorescence Sensing

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“…The apoptosis was evaluated using FITC Annexin V staining in conjunction with propidium iodide (PI) as described previously (Demchenko, 2015). The samples were analysed using FACS Calibor (Becton Dickonson Immunochemistry).…”

Section: Analysis Of Apoptosis By Fitc Annexin V Staining In Conjunct...mentioning

confidence: 99%

How does sperm apoptosis affect the outcome of intrauterine insemination and intracytoplasmic sperm injection?

2022

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Up to 20% of male infertility is caused by abnormal DNA organization of the sperm and anomalies of the sperm apoptosis. The aim of this study was to investigate the sperm DNA apoptosis and viability in patients undergoing intrauterine insemination (IUI) and intracytoplasmic sperm injection (ICSI). In the second part of the analysis, sperm DNA apoptosis and viability were investigated in patients with oligozoospermia and normospermia respectively. A total of 45 IUI and 38 ICSI patients were included in this study. Annexin V analysis was performed to investigate the sperm viability, and TUNEL assay was used to evaluate the sperm DNA apoptosis. Further investigations using 12 oligozoospermia and 11 control samples for sperm viability and sperm DNA apoptosis at different incubation periods and temperatures were performed. The results of this study showed a negative correlation between the sperm DNA apoptosis in IUI patients, but no relationship was observed for the ICSI patients. The second part of this study showed that incubation of semen samples at 37°C for 3 h has detrimental effects on the sperm DNA integrity. In conclusion, the incubation of semen at high temperatures affects the sperm quality. The results of this study showed that these tests can be beneficial for the infertile couples to achieve pregnancy.

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Introduction to Fluorescence Probing of Biological Membranes

Cited by 23 publications

References 112 publications

Sizing Extracellular Vesicles Using Membrane Dyes and a Single Molecule-Sensitive Flow Analyzer

Sizing Extracellular Vesicles Using Membrane Dyes and a Single Molecule-Sensitive Flow Analyzer

Sensing Inside the Living Cells

How does sperm apoptosis affect the outcome of intrauterine insemination and intracytoplasmic sperm injection?

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