10 October 2008
9:24:00 AM
As i mentioned in the earlier post, there are 5 experimental procedures to conduct before vaildifying the hyopothesis. Of the 5, i have only explained soft agar assy, colony assay and western blotting. Here, i will discuss the last 2 experimental procedures.
Immunocytochemistry.
Introduction
Involves plating the cells on a glass slide and subjecting them to specific antibodies , which targets peptide/protein antigens. The bound antibodies would then be detected using a secondary antibody which would cleave flurosense when a thrid party enzyme is introdueced.
Principle
The test stains for darby, aurora-a and beta-tubulin. After the prodcudure, the cells would then we observed under a electron microscope. Darby stains for the chromosome and through the stain you can identify the density of the chromosome within the cells. The location and arrangement of the chromosomes can rougly predict the cell cycle phase the cell is progressing.
Method
Cells are first plated onto the glass slide. Approximately 50000 cells/dish.
1. Dip forceps into ethanol and flame it
2. Using the forceps pick a glass slide and dip into ethanol, flame it until all the residual liquid evaporated.
Caution: Extreme heat may cause the slides to break, hence do not let the glass slide meet the flame directly.
3. Place the glass slide into 30mm dish carefully.
4. Aliquote 1ml of DMEM into each dish.
5. Remove the transfected cultures from the incubator and discard the media
6.Wash the cells in PBS
7. Rinse the cells in trypsin
8. Incubate at 37degrees @5%CO2 for 5minutes
9. Resuspend the cells in 2ml DMEM and transfer into a new 2ml tube.
10. Aliquote 11microL into the haemocytometer and count the cells. Determine the vol of approx. 50000 cells and aliquote the respective amounts into the dishes.
11. Incubate at 37 degrees
The next day, the cells will be blocked with thymidine. Thymidine blocks the cells into the G1/S phase. This is then possible to see the formation of the spindle poles and microtubules after staining. This would allow us to analyze the spindle distance between each other and determine if the knockdown gene has taken any effects. After 22hours, the media will be replaced with fresh DMEM
1. Aliquote 200microL of thymidine into a 50ml tube
2. Aliquote 20ml of DMEM and mix well.
3. Discard old media
5. Replace media with thymidine-DMEM 5ml
6. Incubate for 22hours
After 22hours, the cells are effectively blocked in the G1/S phase and it is possible to carry on to stain the cells.
1. Remove the media
2. Wash in PBS
3. Fix withmethanol for 5mins @ -20degrees
4. Wash with TBS twice
5. Add in 15 BSA/PBS solution for 30mins.
6. Wash with TBS once
7. Incubate with primary antibody for 1hour @ RT
8. Wash with TBS twice
9. Incubate with secondary antibody for 1hour @ RT
10. Wash with TBS once
11. Wash with water once
12. Dry the cover slip and mount using mouting solution onto the glass slide
13. Keep in the dark until use.
The primary antibody used in this experiment is the AIK Rab and Beta-Tubulin Mouse Antibodies. The usage of 2 primary antibodies implys the detection to both the AIK proteins and Beta-Tubulin proteins. The seconday antibodies are Rabb and Mouse respectively as using the same seocndary antibodies might cause confusion later to differentiate the different proteins. The mounting solution is darby stain. Under the electron microscope and viewing under different flourscence, the chromosome arrangement within the cell can be observed, while looking at the darby staining. Under the AIK staining, the microtuble spindle formation can be observed as well. Beta- Tubulin did not flouresence as well and it was difficult to identify beta-tubulin structures in the cells.
In regards to my experiment, this experiment is important to determine if the knock down of the AIP gene would render any side effects to the cell cycle growth and microtubule spindle formation. For example, having an increased distance between the spindle poles may lead to abnormal division of cells. Hvaing decreased distance between the spindle poles may suggest its tendency to form pleuriplody cells.
The spindles of the cells seemed to exisit in the similar range of 10nm, which is normal. Thus it can be concluded that the knockdown of the AIP gene has little or nothing to do with cell growth. This also suggest its potential to induce maligancy is absent as the spindle poles do not hint of any abnormal features.
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