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Analysis of tissue-bound cells: Novel approaches using laser capture microdissection
Matalová, Eva ; Adamová, Eva ; Klepárník, Karel
Recent biomedical research prefers analysis of homogenous samples obtained from primary cells to cell lines. Moreover, attention is paid to cells of low number but high biological impact, such as signalling centres in embryonic development, stem cells or tumour cells. To localize cells of interest within an organ/tissue, histological sections are widely used. However, the methods of choice for further analysis of such samples are mostly limited to histochemistry, immunohistochemistry and in situ hybridization.
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Multi-step synthesis of caspase-3 sensor based on Förster resonance energy transfer
Lišková, Marcela ; 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 Förster Resonance Energy Transfer (FRET) to determine caspase-3 in cell nucleus or cytoplasm by apoptosis for very fast analysis by fluorescence microscopy without sample destroying.
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Senzitive method of Caspase-3 detection in single stem cell
Adamová, Eva ; Klepárník, Karel ; Matalová, Eva
Caspases are involved in physiological process (e.g. cellular differentiation) but also can cause serious disorders. It is getting necessary to develop sensitive, miniaturized and fast methods amenable to analyze small amounts of samples. Luciferin/luciferase chemiluminescence reaction is one of the methods of caspase-3 detection. We have developed a miniaturized device enabling detection of caspase-3 and quantitation just in femtogram level (10–15 g) in single apoptotic human stem cells (neural crest derived). The technology is based on the specific cleavage of modified luciferin by caspase-3, emissions of photons and their detection by photomultiplier tube working in the photon counting regime.
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Sensor based on Förster resonance energy transfer with quantum dot
Lišková, Marcela ; Datinská, Vladimíra ; Klepárník, Karel ; Foret, František
Förster resonance energy transfers (FRET) are based on the nonradiative transfer of energy between a donor and acceptor dye in the distance 1-10 nm. With respect to this, quantum dots (QDs) with a high absorbance can serve as suitable donors of energy in FRET. Here, we present architectures of two sensors based on QD conjugates useful in imaging and diagnostics of cancer. One sensor, based on an oligonucleotide conjugate, can be used for the detection of complementary oligonucletide chains. The other was designed for the detection of enzymes inside cells.
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