Day 29 and 30

After my OGD plates were incubated for 1 and a half hours I treated them with 0.3, 1 and 3uM of TAT and TAT-p110 as follows:


Calculation of the volumes of each of the diluted peptides required to make up final concentration of 0.3 Μm in 500 μl media:

Use the equation V = n/C

For TAT (10mM stock solutions):

To make 0.3μM final: Use the 0.1mM dilution tube, ie. 1/100. 0.1mM is the same as 100μM.
Therefore: 0.3 μM/100 μM = 3x10-3.
3x10-3 x 500 μl = 1.5 μl/500 μl media in ONE WELL. For 15 wells: 1.5 μl / 500 μl x 15 = 22.5 μl/7.5 ml media.
To make 1 μM final: Use 1mM dilution tube, ie. 1/10. 1mM is the same as 1000 μM.
Therefore: 1 μM/1000 μM = 1x10-3.
1x10-3 x 500 μl = 0.5 μl/500 μl media in ONE WELL.
For 15 wells: 0.5 μl / 500 μl x 15 = 7.5 μl/7.5ml media

To make 3 μM final: Use 1mM dilution tube, ie. 1/10. 1mM is the same as 1000 μM.
Therefore: 3 μM/1000 μM = 3x10-3.
3x10-3 x 500 μl = 1.5 μl/500 μl media in ONE WELL. For 15 wells: 1.5 μl / 500 μl x 15 = 22.5 μl/7.5ml media.

For TAT-p110 (4mM Stock solution):
 To make 0.3μM final: Use the 0.04mM dilution tube, ie. 1/100. 0.04mM is the same as 40μM.
Therefore: 0.3 μM/40 μM = 7.5x10-3
7.5x10-3 x 500 μl = 3.75 μl/500 μl media in ONE WELL. For 15 wells: 3.75 μl / 500 μl x 15 = 56.25 μl/2.5ml media

To make 1 μM final: Use 0.4mM dilution tube, ie. 1/10. 0.4mM is the same as 400 μM.
Therefore: 1 μM/400 μM = 2.5x10-3.
2.5x10-3 x 500 μl = 1.25 μl/500 μl media in ONE WELL. For 15 wells: 1.25 μl /500 μl x 15 = 18.75 μl/7.5ml media

To make 3 μM final: Use 0.4mM dilution tube, ie. 1/10. 0.4mM is the same as 400 μM.
Therefore: 3 μM/400 μM = 7.5x10-3.
7.5x10-3 x 500 μl = 3.75 μl/500 μl media in ONE WELL. For 15 wells: 3.75 μl / 500 μl x 15 = 56.25 μl/2.5ml media
After incubating the plates overnight with the drugs i assayed them the next day:
After my assays, I could finally conduct confocal microscopy on my slides! Here is a sneak peak of the confocals from the Leica programme...




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