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Day 20: Analysing the effect of Oxygen glucose deprivation on C17.2 cells upon the treatment of peptides TAT, Myr-p110, and TAT-p110   Aim: to assess the affect of OGD on C17.2 cells after 1.5 hours and 3 hours by assaying LDH, Hoechst and MTT. Reason for assaying LDH: LDH is lactate dehydrogenase which catalyses the conversion of pyruvate -> lactate during anaerobic respiration. Anaerobic respiration only involves the process of glycolysis which occurs only in the cytosol. Thus, if the peptides became cytotoxic to the cells, then the lactate dehydrogenase would leak out into the medium and therefore be detectable by increased absorbance and less transmission. Reason for assaying Hoechst: Hoechst specifically stains nuclei blue. If there are more nuclei present this means that less cells have died as a result of cytotoxicity. If there are less nuclei present this means that more cells have died as a result of cytotoxicity. It is required that we make a blank and then a ...
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Week 4: Inhibiting mitochondrial fission using p110 peptide varients in an attempt to prolong cell survival during neonatal hypoxic ischaemia

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The theory behind the experiment: - There is an increase in mitochondrial mitophagy during neonatal hypoxic ischaemia. The cell becomes stressed as a result of the reduced oxygen in the environment. Reactive oxygen species (ROS) being formed after electrons react with molecular oxygen in the electron transport chain. The mitochondria which have become damaged will undergo the process of mitochondrial fission, mediated by the proteins Drp1, MFF, MID49/51 and Fis1. The mitochondria which have accumulated damage will have their damaged features taken to just one area in the mitochondrion. Once fission has occurred and one mitochondrion has become two, the mitochondrion which had accumulated its damage will undergo mitochondrial mitophagy. - As a result, less ATP is available for the cell but more ROS is produced. In addition, the mitochondrial membrane also becomes leaky releasing apoptosis stimulating factors and cytochrome c, therefore stimulating apoptosis and exaggerating th...
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Day 15: Simply splitting cells I split cells from the two flasks that I left incubating last week and combined them into two flasks but with different media. One flask contained the media that I had previously used before doing my own oxygen glucose deprivation experiments while the other contained a different medium. On Monday (day 16) I will compare the confluence of the cells in both flasks and hopefully will be conducting more cell culture experiments next week!
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