Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Karsten, F.
Establishing Microscopic Assays for Long-Term Live-Cell Imaging of Growth and Proliferation Processes in Cell Populations
[ Diplomarbeit ] Medical University of Graz; 2014. pp. 83
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- Autor*innen der Med Uni Graz:
- Karsten Felix Daniel
- Betreuer*innen:
- Graier Wolfgang
- Malli Roland
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- Abstract:
- Long-term live-cell imaging can be a powerful tool to assess viability, proliferation, and migration of cell populations. Advances over the past couple of decades brought an incubator to the microscope, enabling the observation of cell cultures over days, potentially in combination with flourescence. I established microscopic viability, proliferation, and migration assays on a Zeiss Axiovert Observer and compared it with a cutting-edge system specifically dedicated for those purposes, a Cell-IQ. Three cell lines were tested in those assays, endothelial EA.hy926, cervial cancer HeLa, and pancreatic beta (INS-1) cells, respectively. While the INS-1 cells did not tolerate serum or nutrient deprivation very long, both the EA.hy926 and HeLa cells remained viable in austerity for up to 4 days. This was accompanied by a growth arrest, which could be rescued by re-applying the respective scarce resource after any tested period. Stable knockdown of the Mitochondrial Ca2+ Uniporter (MCU) or Annexin A5 did not influence viability or proliferation of HeLa cells. Transient knockdown of Mitochondrial Ca2+ Uptake 1 (MICU1) did not influence EA.hy926 migration, neither in a scratch nor a cell exclusion zone insert assay. Finally, I correlated intracellular signals determined by Förster Resonance Energy Transfer (FRET)-based sensors with the viability and proliferation of cells. Ca2+ was released from the lumen of the Endoplasmic Reticulum (ER), which was recently shown to correlate with an immediate rise of ATP in this organelle by us. By inducing HeLa cells to proliferate fast or slow by means of cell culture treatments, I found, that the highly proliferative HeLa cells had a significantly elevated Ca2+-coupled [ATP]ER increase compared to the slowly proliferating HeLa cells. Furthermore, glucose-starved HeLa cells had a drastically diminished Ca2+-coupled [ATP]ER increase within minutes, which was restored after about 4 hours of starvation without further treatment. During this time, no change in viability was observed. In my diploma thesis, I compared various analysis techniques, intensity or morphological analysis for viability-, confluency or cell count determination for proliferation-, and manual or automatic migration-assay analysis. Different types of cells, i.e. viable, dead, dividing, and even vacuolized cells could semi-automatically be differentiated by morphological analysis. Based on my results, I conclude that all those assays are feasible for high-throughput analysis.