IADR Abstract Archives
Cytotoxicity and Apoptosis Effect of Newly Designed Florescent Dyes on SCAP
Objectives: The aim of this research is to test the cytotoxicity of new developed florescent dyes. Two types of dyes were checked, one that is specifically connecting to proteins and another that connects to the RNA. The nucleoid acids binding dyes are checked for specificities for RNA and/or DNA. The difference of the structure, charge and the hydrophilicity is expected to influence cellular permeabilisation and cellular toxicity.
Methods: We used mesenchymal stem cells that are isolated from the apical papilla. When cells reached 80–90 % confluence, they were trypsinized (0.05 % trypsin-EDTA, Gibco) (5 min, 37 °C). Further on the cells are transferred into two different vessels. Half of the cells are seeded in 48 well plate and the other half in 25 cm2 plastic flask.
The dyes are administered in the 48 well plate and the cell were immediately placed for observation in InCell Analyzer 6000 (GE HealthCare). In the flasks the florescent dyes were added 24 hours prior the flow-cytometry.
Cellular penetration was observed and recorded on Incell Analyzer 6000 (GE HealthCare) for a period on every 30 minutes for 6 hours.
The differentiation between the apoptotic and alive cells was made by Anexine V kit for flow-cytometry on Navios (BioRad).
For High through-put florescent cellular analysis, the apoptotic cells were visualized with FITC- conjugated anti-anexine V antibody. The nuclei were counterstained with DAPI.
Results: The results show that most of the tested dyes have a significant cytotoxic effect on the cells. Some of the dyes were hydrophilic or negatively charged and could not penetrate the membrane. They were packed in Nano-particles and their specifity and cytotoxicity was evaluated. For some of the tested dyes the was dose dependent.
Conclusions: New specific dyes are urgently needed for understanding cellular processes. Only one RNA specific binding dye is available on the market. Development of new nontoxic dyes discriminating between RNA and DNA or binding specific regions in the proteins will give us valuable tools for understanding the transmembrane transport, RNA translation and protein folding and interactions.
Methods: We used mesenchymal stem cells that are isolated from the apical papilla. When cells reached 80–90 % confluence, they were trypsinized (0.05 % trypsin-EDTA, Gibco) (5 min, 37 °C). Further on the cells are transferred into two different vessels. Half of the cells are seeded in 48 well plate and the other half in 25 cm2 plastic flask.
The dyes are administered in the 48 well plate and the cell were immediately placed for observation in InCell Analyzer 6000 (GE HealthCare). In the flasks the florescent dyes were added 24 hours prior the flow-cytometry.
Cellular penetration was observed and recorded on Incell Analyzer 6000 (GE HealthCare) for a period on every 30 minutes for 6 hours.
The differentiation between the apoptotic and alive cells was made by Anexine V kit for flow-cytometry on Navios (BioRad).
For High through-put florescent cellular analysis, the apoptotic cells were visualized with FITC- conjugated anti-anexine V antibody. The nuclei were counterstained with DAPI.
Results: The results show that most of the tested dyes have a significant cytotoxic effect on the cells. Some of the dyes were hydrophilic or negatively charged and could not penetrate the membrane. They were packed in Nano-particles and their specifity and cytotoxicity was evaluated. For some of the tested dyes the was dose dependent.
Conclusions: New specific dyes are urgently needed for understanding cellular processes. Only one RNA specific binding dye is available on the market. Development of new nontoxic dyes discriminating between RNA and DNA or binding specific regions in the proteins will give us valuable tools for understanding the transmembrane transport, RNA translation and protein folding and interactions.