|
|
The tri-band fluorescent reader
Škrabalová, Denisa ; Čolláková, Jana (referee) ; Dostál, Zbyněk (advisor)
This bachelor thesis deals with the issue of laboratory testing and design of an optical instrument called a tri-band fluorescent reader, which is designed for fluorophores Alexa Fluor 488, Alexa Fluor 555 and Alexa Fluor 647. It contains a theoretical part with key knowledge not only in the natural sciences, such as physics, chemistry, biology, but also the current technical capabilities that are necessary for the practical part. In the practical part is drafting the objectives of the thesis, which are the measurement and evaluation of the samples examined using a monochromator, acquisition and evaluation of images using the existing system, build spectral analysis throughput, create a simulation of custom solutions and create a system engineering design. The mechanical model of the reader assembly was made after measuring the samples and the device and creating the simulation.
|
|
|
Intracellular Calcium Measurement Methods
Číhal, Martin ; Ronzhina, Marina (referee) ; Čmiel, Vratislav (advisor)
This work aims to focus on the issue of intracellular calcium. Not only the theory dealing with the history of fluorescent indicators but also the types of fluorescent indicators and methods of measurements are summarized. The individual steps of measurements on the fluorescent microscope are mentioned as well. There is also created a program for evaluting Ca2+ concentrations in cells made in matlab.
|
|
|
T cells labelling by bimodal contrast agent for in vivo studies of stroke
Krijt, Matyáš ; Poljaková, Jitka (advisor) ; Kříž, Jan (referee)
Stroke is a serious brain injury, which causes sudden death or terminates in permanent neurological disability. Nowadays, tissue plasminogen activator (tPA) is used as the only effective treatment of stroke. One of the potential targets for novel therapy are T cells. Even though the explicit role of T cells in the pathogenesis of brain injury, amounts and timing of all T cell subtypes infiltrating into brain during the stroke still needs further investigation. The research in this field is complicated by the lack of efficient methods for in vivo cell tracking. Therefore the aim of this thesis was to develop a method of T cells labelling by MRI contrast agent in order to investigate T cells distribution in ischemic mice model using in vivo MR imaging. T cells were isolated from C57/BL6 mice in two step isolation protocol using gradient centrifugation and magnetic separation with the efficiency of 97 %. The isolated cells were labelled with 100 μg Fe/mL of Molday ION Rhodamine B contrast agent. The labelling efficiency after 17 hours of cells incubation was higher than 99 %. The labelled cells were cultured with CD3 and CD28 antibodies resulting into the 74 % viability of labelled T cells compared to 83 % viability of non labelled T cells. The labelled T cells were visualized by fluorescent...
|
|
| |
|
| |
|
|
The tri-band fluorescent reader
Škrabalová, Denisa ; Čolláková, Jana (referee) ; Dostál, Zbyněk (advisor)
This bachelor thesis deals with the issue of laboratory testing and design of an optical instrument called a tri-band fluorescent reader, which is designed for fluorophores Alexa Fluor 488, Alexa Fluor 555 and Alexa Fluor 647. It contains a theoretical part with key knowledge not only in the natural sciences, such as physics, chemistry, biology, but also the current technical capabilities that are necessary for the practical part. In the practical part is drafting the objectives of the thesis, which are the measurement and evaluation of the samples examined using a monochromator, acquisition and evaluation of images using the existing system, build spectral analysis throughput, create a simulation of custom solutions and create a system engineering design. The mechanical model of the reader assembly was made after measuring the samples and the device and creating the simulation.
|
|
|
T cells labelling by bimodal contrast agent for in vivo studies of stroke
Krijt, Matyáš ; Poljaková, Jitka (advisor) ; Kříž, Jan (referee)
Stroke is a serious brain injury, which causes sudden death or terminates in permanent neurological disability. Nowadays, tissue plasminogen activator (tPA) is used as the only effective treatment of stroke. One of the potential targets for novel therapy are T cells. Even though the explicit role of T cells in the pathogenesis of brain injury, amounts and timing of all T cell subtypes infiltrating into brain during the stroke still needs further investigation. The research in this field is complicated by the lack of efficient methods for in vivo cell tracking. Therefore the aim of this thesis was to develop a method of T cells labelling by MRI contrast agent in order to investigate T cells distribution in ischemic mice model using in vivo MR imaging. T cells were isolated from C57/BL6 mice in two step isolation protocol using gradient centrifugation and magnetic separation with the efficiency of 97 %. The isolated cells were labelled with 100 μg Fe/mL of Molday ION Rhodamine B contrast agent. The labelling efficiency after 17 hours of cells incubation was higher than 99 %. The labelled cells were cultured with CD3 and CD28 antibodies resulting into the 74 % viability of labelled T cells compared to 83 % viability of non labelled T cells. The labelled T cells were visualized by fluorescent...
|
|
|
Preparation of analogues of MRI contrast agents and study of their internalization on cell cultures
Krijt, Matyáš ; Poljaková, Jitka (advisor) ; Martínková, Eva (referee)
Magnetic resonance imaging (MRI) is a commonly used non-invasive method to examine internal organs and soft tissues. In order to increase signal intensity and improve specificity contrast agents (CAs) have been developed, which affect T1 or T2 relaxation times. T1-contrast agents are mostly based on Gd(III) complexes. The aim of this thesis was to prepare analogues to commercial Gd(III)-CAs by changing the central atom for europium and use europium fluorescence properties for observing CAs' cell-distribution. However, due to strong cell auto-fluoresce the fluorescence of complexes did not provide convincing images reporting their biochemical fate. The toxicity of prepared complexes was tested on adherent cell cultures and evaluated by Fluorescence-Activated Cell Sorter (FACS). Internalization of the CAs' analogues was confirmed also by elemental analysis (ICP-MS). (In Czech)
|
|
|
Intracellular Calcium Measurement Methods
Číhal, Martin ; Ronzhina, Marina (referee) ; Čmiel, Vratislav (advisor)
This work aims to focus on the issue of intracellular calcium. Not only the theory dealing with the history of fluorescent indicators but also the types of fluorescent indicators and methods of measurements are summarized. The individual steps of measurements on the fluorescent microscope are mentioned as well. There is also created a program for evaluting Ca2+ concentrations in cells made in matlab.
|
guest ::
