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Cell Viability Measurement
Pelc, Pavel ; Janoušek, Oto (referee) ; Čmiel, Vratislav (advisor)
This work discusses the viability of cells. It describes the principle of fluorescence, fluorescent methods, fluorescent microskopy and the method of dyeing of the cells with a special kit that contains calcein AM and ethidium homodimer dyes. Further it describes the program for analysis and evaluation of viability on two different cellular cultures.
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Cell Viability Measurement
Prokopyeva, Elena ; Sekora, Jiří (referee) ; Čmiel, Vratislav (advisor)
This master's thesis deals with the ways of cell viability determination and its measuring. The first part of this thesis has a brief introduction into the theory of cell viability, also the methods of cell viability measurement and different types of fluorescent probes are described. The thesis further includes a brief description of the image processing methods, as well as the fluorescent microscope and applied filters are described. In the practical part the system implemented at a graphical programming environment LabVIEW used for the cell viability evaluation is described, functions of applied units are explained. The program is checked on model data.
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Viability evaluation on cultivated mezenchymal cells
Jehličková, Jana ; Odstrčilík, Jan (referee) ; Čmiel, Vratislav (advisor)
This thesis deals with the possibilities of cell viability determination and its evalution. The thesis in theory describes cell cultivation, different methods of staining cells and display options. In the practical part is mentioned the preparation of the observed experiment and there are shown the examples of cell images created by confocal microscopy. Additionaly there are created the data analysis algorithm in Matlab. For setting suitable analysis parameters is formed user interface.
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Comparison of in vitro methods for the study of cytotoxicity
Eliášová, Pavla ; Maixnerová, Jana (advisor) ; Bárta, Pavel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Pavla Eliášová Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Comparison of in vitro methods for the study of cytotoxicity People are exposed to a growing number of toxic substances from the environment. Endocrine disrupting chemicals (EDCs) are a broad category of molecules that are thought to cause adverse effects on the endocrine system by interfering with the synthesis, transport, degradation or action of endogenous ligands. One of the aims of this thesis was to determine the in vitro toxicity of 17 selected endocrine disruptors on the human hepatocellular carcinoma HepG2 cell line. Cell viability was determined using the CellTiter96® AQueous One Solution Cell Proliferation Assay colorimetric method, the principle of which is the reduction of MTS to the colored product formazan by mitochondria in viable cells. The cytotoxic potential of the compounds was expressed by using the toxicological parameter IC50, which was measured in three time intervals (6, 12 and 24 hours). For 14 substances: Atrazine, DHEP, Bisphenol A, Carbofuran, 3-hydroxycarbofuran, Cypermethrin, DDE, DES, MEHP, PCB 118, PCB 153, PFOA, PFOS, Propiconazole, IC50 > 100 µM (respectively > 250 µM) was...
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Induction of oxidative stress in skin keratinocyte cells
Martínková, Nikola ; Jirkovská, Anna (advisor) ; Hradiská Breiterová, Kateřina (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Student: Nikola Martínková Supervisor: PharmDr. Anna Jirkovská, Ph.D. Consultant: Mgr. Petr Jošt, Ph.D. Title of diploma thesis: Induction of oxidative stress in skin keratinocyte cells Keratinocytes are part of the epidermis and represent the majority of cells in the upper layer of human skin. When these cells are damaged by mutagenic substances, changes in the composition of the genetic material may occur, and even cell death may occur. Such substances that induce changes in DNA and have mutagenic potential include the chemical warfare agent sulfur mustard. The purpose of this study was to compare the HaCaT keratinocyte cell line with the experimentally derived 3HSM4 cell line in response to hydrogen peroxide-induced oxidative stress. The 3HSM4 cells were prepared by selecting resistant clones of HaCaT cells that were repeatedly exposed to the cytotoxic effects of sulfur mustard. The results of the comparison show that 3HSM4 cells proved to be more resistant to hydrogen peroxide. When glutathione (GSH) synthesis was blocked in both cell types, the cytotoxic effect of hydrogen peroxide was the same. Although 3HSM4 cells were shown to be more resistant in terms of cytotoxicity, they were found to have much...
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In vitro cytotoxicity study of selected antimycobacterial substances on human hepatocytes
Klimová, Miroslava ; Kuchařová, Monika (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Student: Miroslava Klimová Supervisor: Mgr. Monika Kuchařová, Ph.D. Consultant: Mgr. Pavel Bárta, Ph.D. Title of diploma thesis: In vitro cytotoxicity study of selected antimycobacterial substances on human hepatocytes Clinical research is constantly progressing, new, missing drugs are being searched for, more effective substances are being synthesized, adverse effects are being minimized. The aim of this thesis was to determine the effect of ten substances (JZ195, JZ186, MH17, JZ178, MH9, MH18, JZ177, JZ170, JZ172, JZ174) in the HepG2 cell line and evaluate their cytotoxic potential. The tested substances were derived from the structure of pyrazine-2- carboxamide and synthesized within the Department of Organic and Bioorganic Chemistry at the Faculty of Pharmacy in Hradec Králové, Charles University and developed for the purpose of having promising effect on mycobacterial strains. Cytotoxicity was evaluated in vitro by CellTiter96® AQueous One Solution Cell Proliferation Assay, commercially available colorimetric method based on reduction of the tetrazolium salt to a violet-coloured formazan which directly reflects the number of viable cells. We assessed the hepatotoxic effect using the IC50...
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Study on cytotoxicity of compounds in vitro.
Vašková, Lucie ; Maixnerová, Jana (advisor) ; Kuchařová, Monika (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Studentka: Lucie Vašková Školitel: RNDr. Jana Maixnerová, Ph.D. Název diplomové práce: Study on cytotoxicity of compounds in vitro The subject of this diploma thesis was to assess the effect of newly synthesized antimycobacterial substances on the viability of human hepatocellular carcinoma (HepG2) cells. The tested substances were esters (HE-nMe, HE-4PHOPH, HE-KARVA, HE-2NAFT, HE-METRO, HE-CH2PY, HE-8CHIN) and thioesters (HES-4H, HES- nETH) of antituberculotic isoniazid. Experiments performed with these substances have shown, that like isoniazid, the substances inhibit InhA enzyme in mycobacteria and therefore interfere with cell wall biosynthesis. Isoniazid is a drug standardly used in the first line of TB treatment. Together with other first-line antituberculotics, some hepatotoxic potential has been reported during treatment. To assess the possible cytotoxic effect of the tested isoniazid derivatives, the standard human hepatocyte cell line HepG2 was chosen as the cell model. Cell viability was assessed by a colorimetric method that measures the metabolic activity of cells based on the reduction of the tetrazolium compound MTS. Obtained values were quantitatively compared using the toxicological...
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Utilization of tissue cultures for toxicology of the environment.
Polanská, Daniela ; Cajthaml, Tomáš (advisor) ; Klusoň, Petr (referee)
5 Abstract Five substances from the group of so-called personal care products, known for their low degradability and regular environmental detection, were tested for toxicity using two fish tissue lines (RTgill-W1 a RTG-2) isolated from rainbow trout (Oncorhynchus miykiss). The tested substances were hexadecylpyridinium chloride (HDP), chlorhexidine (CHX), octenidine (OCT), thymol (THM) and triclosan (TCS). A cell viability assay was performed with each of these compounds using Alamar Blue ™ (AB), 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) and neutral red (NR) protocols. The results were used to construct dose-response curves along with an EC50 value for each of these substances. The EC50 values ranged from 0,51 (HDP) to 33,75 µg.ml-1 (THM) for RTgill-W1 and from 0,31 (HDP) to 33,37 µg.ml-1 (THM) for RTG-2. The theoretical LC50 estimation was calculated according to Tanneberger et al. (2013). For all substances, cytochrome P450 1A activity was monitored using 7-ethoxyresorufin-o-deethylase (EROD), four out of five tested chemicals were statistically positive for EROD, the highest EROD response was observed for the most toxic compound - HDP. Only TCS did not show statistically significant cytochrome P450 1A activity. In addition, oxidative stress was measured with the fluorescent dye...
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