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The fluorescent marking of glass microelectrodes for the patch clamp system
Klepáčová, Ivana ; Ronzhina, Marina (referee) ; Chmelíková, Larisa (advisor)
The paper deals with the issue of the fluorescent marking of glass microelectrodes for the patch clamp system. It analyses in more details the possibilities of creation of glass microelectrodes from the choice of appropriate glass type to the construction of a microelectrode, which can be used in practice in experimental measurements. Phases of creation process of glass microelectrodes document possibilities of the development of the given technique. The description of fluorescence phenomenon includes the classification and description of fluorophores. The paper contains a theoretical analysis of the given issues and presents it in its comprehensive form. The practical part of the thesis suggests the procedure of the external fluorescent marking of glass microelectrodes which can be used in the measurement by means of the patch clamp system. It is concerned with the testing of the established procedure and provides the evaluation of the possible application of the suggested procedure in practice.
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Simulation of electrophysiological experiments
Kubáček, Svatopluk ; Provazník, Ivo (referee) ; Rychtárik, Milan (advisor)
Simulation of electrophysiological experiments enables to gain valuable data about bioelectrical processes occurring on isolated cells. This thesis deals with basic principals allowing the creation of bioelectrical phenomenons and measuring techniques that need to be used in order to collect information about these phenomenons. The outcome of the thesis is a programme that serves as an instrument for simulation of measuring techniques applied on Luo-Rudy model. The programme contains basic measuring techniques – current clamp and voltage clamp – providing the possibility to change basic parameters of the model. The results of this work are later compared with the accessible literature.
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The effect of cryopreservation on the current response of CaV 3.1 transfected HEK293 cells
Svoboda, O. ; Fohlerová, Z.
The main disadvantage of long-term storage of living cells in liquid nitrogen is its relatively fast evaporation. For this reason, it is necessary to refill Dewar flask quite often. In our experiments, we used low temperature freezer (-80 °C) as more economical option to liquid nitrogen and we would like to show that there is no significant influence of long-term storage of mammalian cell-sat a low temperature. The effect of temperature was studied as the electrophysiological characteristic of the whole-cell Ca2+ current in HEK293 cells. The current responses were measured from cells stored for eight months at -80 °C and compared with previously published papers. The current traces and I-V curves showed that there are no changes in current response between cells frozen at a low temperature and previously published results. From our results can be concluded that the low temperature freezer is an adequate option for storage of mammalian cells for a couple of months.
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Patch Clamp Technique – The Tool for Ions Transport across Model and Real Biological Membranes
Navrátil, Tomáš ; Jaklová Dytrtová, J. ; Jakl, M. ; Šestáková, Ivana ; Mareček, Vladimír ; Krůšek, Jan
The project is aimed at the preparation and characterization of model and/or real phospholipid bilayers at stabilized phase boundaries enabling studies of transfer of charged and uncharged species between the two phases. The best way for investigation of inorganic ion transfer is provided by using mercury as one of the phases, in view of the large amount of the relevant electrochemical data available, whereas the transfer of more complex species is better studied in the system of an organic and an aqueous phase stabilized by (agar) gel. The results contribute to the understanding of ion transport across real membranes. Polypeptides and peptides capable forming ionic channels and enabling transport of various ions and molecules across the membranes, will be incorporated into the model membranes. The possibilities of transport of selected compounds across the model membrane will be investigated using cell permeable peptides as well.
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