Národní úložiště šedé literatury Nalezeno 6 záznamů.  Hledání trvalo 0.00 vteřin. 
Parallel single-cell analysis of active caspase-3/7 in apoptotic and non-apoptotic cells
Ledvina, Vojtěch ; Klepárník, Karel
Caspases are proteases that play key role in the process of apoptosis, the programmed\ncell death. Among them, caspase-3 and -7 are main executioner caspases that cleave\nmany vital proteins during apoptosis and after their widespread activation, the process\ncannot be reversed. To analyze caspase-3/7 activation within single cells, a miniaturized\ndevice for parallel analysis of eight samples was developed. The assay is based on the\nmodified luciferin-firefly luciferase bioluminescence (BL) system. Individual\nsuspended cells were collected and transferred into detection microvials using a\nmicromanipulator. The bioluminescence was detected using a photon counting head\nwith cooled photcathode. The LOD suitable for detection of active caspase-3/7 in both\napoptotic and non-apoptotic cells was reached.
Characterization of FRET sensor
Datinská, Vladimíra ; Klepárník, Karel ; Belšánová, B. ; Minárik, M. ; Foret, František
In this study, we present characterization of sensor based on Fӧrster resonance energy\ntransfer (FRET). The sensor is composed of ssDNA chain attached to a laboratory\nsynthesized quantum dot (QD). A complementary chain of a sample is labeled by a\nluminescent dye. When the dsDNA hybrid is formed, the energy from the QD (donor)\nis transferred to the dye (acceptor) and FRET is observed as a decrease of QD\nluminescence emission intensity and an increase of dye luminescence emission\nintensity.
Synthesis and analysis of quantum dot conjugates intended for fret sensor
Datinská, Vladimíra ; Klepárník, Karel ; Belšánová, Barbora ; Minárik, M. ; Foret, František
Our work is focused on synthesis of sensor based on Fӧrster resonance energy transfer (FRET), which is aimed to be used for DNA mutation detection. The sensor is based on the attachment of laboratory synthesized quantum dot (QD) to ssDNA. A complementary chain of a sample is labeled by a fluorescent dye. Thus, only if dsDNA between both complementary chains is formed, the energy from QD (donor) can be transferr ed to the dye acceptor) and FRET is observed.
Simple route of caspase-3 FRET sensor synthesis using “click chemistry”
Lišková, Marcela ; Křenková, Jana ; Klepárník, Karel ; Pazdera, P. ; Foret, František
Programmed cell death or apoptosis is regulated process of cell suicide. The central role in apoptosis play cysteine proteases called caspases. Caspases recognize tetra-peptide sequences Asp-Glu-Val-Asp (DEVD) on their substrates and hydrolyze peptide bonds after aspartic acid residues. Various techniques for the determination of caspase-3 are commercially available e.g. Enzyme Linked Immuno-Sorbent Assay (ELISA), Western blotting or flow cytometric analysis. The products of the cleavage can be detected by spectrophotometry, fluorimetry, chemiluminescence (CL) or ELISA. In this work, we suggested fluorescent sensor based on easily prepared Förster Resonance Energy Transfer (FRET). We use very simple chemistry called “click”. This type of chemistry takes advantages of quickness, simplicity and cheapness. “Click chemistry” is based on usage of various functional group and cross-linkers to combine individual molecules together.
Quantum dot-based Fӧrster resonance energy transfer bioanalysis
Datinská, Vladimíra ; Klepárník, Karel ; Belšánová, B. ; Minárik, M. ; Foret, František
We describe a new configuration for Fӧrster resonance energy transfer (FRET) between a quantum dot (QD) donor and a fluorescence dye acceptor attached to DNA molecule. This FRET sensor is aimed to be used in genomic analysis for DNA mutation detection applicable especially in cancer research.
Sample preparation for single cell analysis
Adamová, Eva ; Basova, E. Y. ; Potáčová, Anna ; Foret, František ; Matalová, Eva ; Klepárník, Karel
Development of sensitive, miniaturized and fast methods aims to reliable analyze even small amounts of samples. Micromanipulation systems such as laser capture microdissection enable to collect just single cells from cell suspensions or tissue samples. One option for further analysis is the bioluminescence reaction based on luciferin/luciferase conversion. This approach was used for detection of active caspases, caspase-3/7. Droplets based microfluidic systems provide benefits in biological, pharmaceutical and chemical research.

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