Day 9: Oxygen/glucose deprivation to C. 17 cells

We wanted to observe the effect of oxygen glucose deprivation on the C 17 cells. The way in which we did this was by immersing the cells in artificial cerebrospinal fluid. The artificial cerebrospinal fluid did not contain any glucose and treated it with a vaccum to ensure that there was no oxygen in the fluid. we incubated two plates for 1 hour and a half and another two for 3 hours by placing it in a hypoxia chamber and flooding the chamber with nitrogen.

The result on the control plates:


The control cells did not under go oxygen glucose deprivation. The top photo shows the neurones with no marker dyes whilst the bottom shows that they were treated with markers JC1 and Hoechst. Hoechst stains the nuclei and chromatin. JC1 is a special fluorescent dye that changes colour depending on membrane potential. When the mitochondrial membrane potential is high, JC1 is aggregated and fluoresces red. However when the mitochondrial membrane potential is low, the JC1 forms monomers and fluoresces green.

In healthy mitochondria, the membrane potential is high and when mitochondria are stressed the mitochondrial membrane potential decreases, indicating that mitophagy may follow. The control cells shows that there was a substantial amount of red fluorescence indicating that the mitochondria were not stressed and that JC1 was still in its aggregate form.

 Upon oxygen/glucose deprivation:


:


 There is significantly less red fluorescence in the plates exposed to oxygen glucose deprivation, indicating that the overall mitochondrial membrane potential was reduced.

Comments

Post a Comment

Popular posts from this blog

Week 4: Inhibiting mitochondrial fission using p110 peptide varients in an attempt to prolong cell survival during neonatal hypoxic ischaemia

Image
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
Read more
Image
Day 23, and 24 I split the cells which were present in the flask into two separate flasks for more examination next week on different experiments. After counting the cells, I found that there were 1.124 x 10 5 /ml cells were found in the cell sample and 350K cells were required in each of the 6 plates. Calculation for the volume of cells and media needed in each plate: 350, 000 x 8 = 2, 800, 000. 2, 800, 000/1, 124, 000 = 2.45 ml cells are required in the media ie. 2.5 ml cells. 3ml media is required in each plate, therefore 3ml x 8 = 24 ml media 1.124 x 10 5 /ml cells were found in the cell sample and 350K cells were required in each of the 6 plates. I plated the cells as follows: - I placed 3ml media in each of the plates, incubated them to allow them to settle for atleast 4 hours before the oxygen glucose deprivation. After 4 hours: - Harvest the No OGD and place it in its own epindorph tube. Leave plate 3 in the incubator because that needs to be harvested after
Read more