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ePaper created 2017-06-21, 10:00:36 | version 1.38.0
temperature-related damage of the DPSC study | Discussion The first indications to a temperature-related damage of the DPSC were seen in the Live/Dead Assay. Calcein is able to penetrate the membrane and is only converted to a fluorescent colouring agent inside of an intact cell. If the cell membrane becomes permeable as a result of damages, calcein will not remain inside the cell. As a consequence, Ethidium-homodimer-D1 will enter the cell in ex- change. This substance is not permeable for intact membranes and will fluoresce red when combined with DNA. Interestingly significant thermally-induced dam- ages were only observed at temperatures ranging from 46.5 °C ± 0.5 °C. Starting at this temperature, cell membranes are destroyed apparently. Temper- atures from 56.5 °C ± 0.5 °C form another threshold at which the 50 % lethality limit was reached. If the vitality test was conducted 24 h after ther- mal treatment, almost twice as much lethal cells as observed 1 h after incubation were seen at tem- peratures from 46.5 °C to 56.5 °C. It appears that repairing processes cannot eliminate the thermal damage. Contrarily, thermal treatment will result in a lethal reaction even 1 h later. Fig. 6: TEM: Control cells at 37 °C. K: Nucleus; ER: endoplasmatic reticulum, RER: rough endoplasmatic Fig. 6 reticulum; M: mitochondria; Z: cytoskeleton; arrows: markers of the nuclear membrane. Starting at 56.5 °C, most cells died immediately, probably due to denaturation of the proteins (co- agulation). Usually, a temperature level of 62 °C is given as the starting point for coagulation in the literature. However, the Live/Dead Assay does not allow any conclusions on the effects of the damages on the cell organells, compartments or physiological reactions such as protein production. Consequently, HSP tests and electron microscopic examinations of the ultrastructure were conducted additionally. Heat Shock Proteins (HSP) were detected very well at 50 °C by an antibody reaction. The cells were distinctly coloured, which implies a significant re- action of the cell on the temperature-related stress. These cells were still able to synthesise the proteins and to survive for some time. Controls only showed only a light colouring, which may be the result of an unspecific reaction of the antibody with different Fig. 7: TEM: Due to thermal treatment at 50 °C, the cells are rounded and the cell membrane forms vesicles (left). Mitochondria exhibit a disrupted structure of the christae, while the nuclear plasma starts to condense and the nucleus (K) itself often appears to be uncharacteristically flapped (arrow). The nuclear membrane (*) seems to be partially inflated or dissolved. Fig. 7 roots 2 2017 35