M&M

3. Materials & Methods

3.1 Materials
Apart from ADP, Thrombin and Aspirin which have been explained before, further substances are required as well which would be described below.




3.1.1 HEPES buffer
HEPES buffer is a suitable alternative general-purpose zwitterionic buffer. HEPES buffer does not bind to Mg +2, Ca +2, Mn +2 and Cu +2. HEPES buffer is one from ten important biological buffers.
-HEPES buffer with CaCl2 is used in this experiment .It is a zwitterionic buffer does not bind Ca +2 in plasma. HEPES buffer can exhibit toxicity if the concentration is greater than 40 mM .20 mM HEPES is the most satisfactory concentration. HEPES buffer can maintain pH. That is why it is more used in cell culture.
HEPES used in this experiment is consisting of:
-10 mM NaCl, 10mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
,2.7 mM CaCl2 ,5 mM Glucose ,0.5 mg/mL BSA ,Distilled water up to 500mL ,Few drops of NaOH to reach the pH to 7.4 (Lepe-Zuniga JL et al , 1987)

3.1.2. Fixative solution
Fixative solution used in this experiment is formaldehyde also known as methanal, CH2O MW=30.03, which is a very good stabiliser and fixative in biological studies. Usually it is used in 4% w/v concentration. It does limit oxidation and polymerisation.

3.2. Method

3.2.1. General description
The idea if planning this method is based on similar study conducted by Jef L and colleagues in 2006.
In a nut shell the over view of all done in the method of this experiment is:

1-Preparing HEPES buffer
2-Pareparting PRP: Platelets Rich Plasma (Centrifuging in 200 g for 10 minutes)
3-Prepraing series of dilution of Thrombin and ADP.
4-Adding Alcohol (Ethanol) [tubes 1 to 8] and adding Aspirin [tubes 9 to 16]
5- Taking 10µL from indicated tubes +90µL fixative (formaldehyde)
6-Taineg 1ml from above and mixing with 1 ml distilled water.
7-Flowcytometry to plot a histogram and gather the data.
More details comes bellow.

3.2.2 Platelet isolation
First stage is platelets isolation.10 mL blood was collected in 2 mL anticoagulant ACD (anticoagulant citrate dextrose solution) (2.5% sodium citrate, 1.5% citric acid, and 2% glucose).The donor should not take Aspirin within last few days. Then the specimens are centrifuged for 10 minutes in 2000g to reach the Platelet Reach Plasma (PRP). Follow by order HEPES buffer is prepared to use in next stages as the main buffer of this experiment.

3.2.3 Solution A, Solution B
Two separate solutions called solution A and B are prepared as follow:
-Solution A: Solution A is consist of: 2mL buffer + 50 µL PRP + 2µL Alcohol (Ethanol) Concentration of Ethanol = 0.01 % (0.00009746) V/V
-Solution B: Solution B is consist of: 2mL buffer + 50 µL PRP + 2µL Aspirin
Concentration of Aspirin=0.01 % (0.00009746) V/V

3.2.4 Preparing preliminary concentration of Thrombin and ADP
A set of 16 tubes are required. The following concentration of Thrombin and ADP are made. By the end of this stage the concentration of Thrombin and ADP content of each tube will be as the following table.















Table 3.2.4.1. Preliminary concentrations of Thrombin and ADP.

Tube 1
10 µL buffer
Tube 2
10 µL buffer
Tube 3
0.10 Unit/mL Thrombin
Tube 4
0.30 Unit/mL Thrombin
Tube 5
1.00 Unit/mL Thrombin
Tube 6
3.00 Unit/mL Thrombin
Tube 7
10.00 Unit/mL Thrombin
Tube 8
200µM ADP
Tube 9
10µL buffer
Tube 10
10µL buffer
Tube 11
0.10 Unit/mL Thrombin
Tube 12
0.30 Unit/mL Thrombin
Tube 13
1.00 Unit/mL Thrombin
Tube 14
3.00 Unit/mL Thrombin
Tube 15
10.00 Unit/mL Thrombin
Tube 16
200µM ADP

By the end of this stage 16 tubes are ready. Now extra 16 tubes are required to carry on the experiment to prepare the final concentrations of Thrombin and ADP.


3.2.5. Preparing final concentrations of Thrombin and ADP
In these series of tubes we need to take 10 µL from the previous tubes and mix with 90 µL of solution A for tubes 1 to 8 and solution B for tubes 9 to 16. Hence we will see the new set of tubes in which the concentration of thrombin and ADP will become 10 fold less. By the end of this stage in new set of tubes the concentration of Thrombin or ADP and content of each tube will be as the following table.




Table 3.2.5.1. Final concentrations of Thrombin and ADP.

Tube 1
10mL buffer
Tube 2
10mL buffer
Tube 3
0.01 Unit/mL Thrombin
Tube 4
0.03 Unit/mL Thrombin
Tube 5
0.10 Unit/mL Thrombin
Tube 6
0.30 Unit/mL Thrombin
Tube 7
1.00 Unit/mL Thrombin
Tube 8
20µM ADP
Tube 9
10mL buffer
Tube 10
10mL buffer
Tube 11
0.01 Unit/mL Thrombin
Tube 12
0.03 Unit/mL Thrombin
Tube 13
0.10 Unit/mL Thrombin
Tube 14
0.30 Unit/mL Thrombin
Tube 15
1.00 Unit/mL Thrombin
Tube 16
20µM ADP

After this stage we need to wait for ten minutes for each tube .Then Annexin V will be added.

3.2.6. Adding Annexin V and preparing for flowcytometry
At this stage 5µL Annexin V is added to each tube. Annexin V is labelled with fluorescent substance (florin). Then after 50µL of each tube is taken and mixed with 100 µL fixative (formaldehyde) in another tube. We can now leave the tubes for a couple of hours. It is very important to do the flowcytometry at the same day. Although they are fixed with fixative but they should not be kept for more than some hours. Before final stage, 1 mL of each tube is taken and mixed with distilled water in another series of tube. This is to prepare for flowcytometry.



3.2.7. Flowcytometry
The final stage is flowcytometry. The flowcytometry machine will be set as described above and the results would be gathered. The results of Flowcytometry come in next part.