Oswald Hope Robertson
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Oswald Hope Robertson (2 June 1886 – 23 March 1966) was an English-born medical scientist who pioneered the idea of blood banks in the "blood depots" he established in 1917 during service in France with the US Army Medical Corps.
Robertson was born in Woolwich in south-east London, but at the age of one-and-a-half he emigrated with his parents to California, settling in the San Joaquin Valley. He attended local schools in Dinuba, then graduated from the Polytechnic High School in San Francisco.
His initial plan to study basic biology was changed by a meeting with an American medical student while on holiday in Germany. After attending some lectures on anatomy, he decided to study medicine, being admitted to the University of California in 1906. He later studied at Harvard Medical School, the Massachusetts General Hospital and the Rockefeller Institute for Medical Research, but had to cut short his studies during World War I when he was called to join medical teams in France. Here he experimented with preserving human blood cells for use in blood transfusions, and became recognised as the inventor of the blood bank.
[edit] Commemoration
Friday, October 23, 2009
Thursday, April 23, 2009
Conslusion
As already seen in the discussion part, the hypothesis which expressed there is no relationship between activation/inhibition of the platelets and externalization the phosphatidylserine would be, based on the results, ruled out.
Another hypothesis which expresses the relation ship is still on the table. It is not possible to declare:
-Activation of the platelets by Thrombin causes externalization of phosphatidylserine on the surface of the platelets.
-In this experiment the same results could be not applied to ADP as the test was performed for different concentrations of ADP but satisfactory results were not reached. Changing method in this regard in suggested.
-Inhibition of the platelets by Aspirin causes internalization of phosphatidylserine on the surface of the platelets. This statement has been approved by comparing the results of controls with and with out Aspirin.
In addition, having seen the papers, we would realize that COHEN Zoë and colleagues conducted a research in 2004 and their find out declare that if platelets become activated, they would express phosphatidylserine on the outer leaflet of the plasma membrane. They have working with ADP as they say ADP-induced PS exposure. They wanted to realise the role of Caspase in this process as well, while we were not. It can be a suggestion for further works as well to consider different enzymes in this process. Their findings could be useful to prove the activatory role of ADP as well.
Finally, the characteristics of Annexin V have been approved again. The property which they bind with negatively charged phospholipid on the surface of the cells, although it is not the main statement of this experiment but this viable facts could be used to be applied in other experiments as well.
6.1. Suggestions for further works
It should be realised how Thrombin/ADP or Aspirin apply their changes on the platelets. If we were working on cells which have nuclei the possibility of effect on the nuclei could be mentioned. But the platelets which are circulating in blood are not same cell. Therefore changing the expression of a gene which is highly possible for physiological reactions is ruled out. The other and simple way which comes to mind is acting by the aid of a second messenger. Further studies should be based on understanding the mechanism of this effect.
Repeating this experiment with different activators is suggested as well, in particular due to unclear results from ADP.
Further experiments should investigate about the role of Caspase and try to find out the mechanism of inhibition and activation with more details.
As stated about the receptors of ADP and Thrombin. The mechanism of action of stimulation (activation or inhibition) should be considered to find more relationship between the factor of change and response.
Another hypothesis which expresses the relation ship is still on the table. It is not possible to declare:
-Activation of the platelets by Thrombin causes externalization of phosphatidylserine on the surface of the platelets.
-In this experiment the same results could be not applied to ADP as the test was performed for different concentrations of ADP but satisfactory results were not reached. Changing method in this regard in suggested.
-Inhibition of the platelets by Aspirin causes internalization of phosphatidylserine on the surface of the platelets. This statement has been approved by comparing the results of controls with and with out Aspirin.
In addition, having seen the papers, we would realize that COHEN Zoë and colleagues conducted a research in 2004 and their find out declare that if platelets become activated, they would express phosphatidylserine on the outer leaflet of the plasma membrane. They have working with ADP as they say ADP-induced PS exposure. They wanted to realise the role of Caspase in this process as well, while we were not. It can be a suggestion for further works as well to consider different enzymes in this process. Their findings could be useful to prove the activatory role of ADP as well.
Finally, the characteristics of Annexin V have been approved again. The property which they bind with negatively charged phospholipid on the surface of the cells, although it is not the main statement of this experiment but this viable facts could be used to be applied in other experiments as well.
6.1. Suggestions for further works
It should be realised how Thrombin/ADP or Aspirin apply their changes on the platelets. If we were working on cells which have nuclei the possibility of effect on the nuclei could be mentioned. But the platelets which are circulating in blood are not same cell. Therefore changing the expression of a gene which is highly possible for physiological reactions is ruled out. The other and simple way which comes to mind is acting by the aid of a second messenger. Further studies should be based on understanding the mechanism of this effect.
Repeating this experiment with different activators is suggested as well, in particular due to unclear results from ADP.
Further experiments should investigate about the role of Caspase and try to find out the mechanism of inhibition and activation with more details.
As stated about the receptors of ADP and Thrombin. The mechanism of action of stimulation (activation or inhibition) should be considered to find more relationship between the factor of change and response.
Discussion
The hypothesis of this project as stated before is mainly about finding a relationship between activators of platelets (Thrombin and ADP) and Inhibitor of palettes (Aspirin) on externalisation the phosphatidylserine on the outer membrane of the platelets which would bind to Annexin V labelled with fluorescent. Therefore by the end of running the experiment for five times the data should be analysed to find logic relation ship between aforementioned parameters. By comparing the result of flowcytometry, few groups of analysis are reached. After ward in a separate graph they are compared. In all charts Y error bars represent Standard Deviations.
5.1. Thrombin and Aspirin
5.1.1. The first chart will demonstrate the externalisation of the Thrombin in different concentrations. It is with out Aspirin .This chart would reveal that what is the effect of Thrombin in externalisation of Phosphatidylserine with out Aspirin.
Graph 5.1.1.1. Externalising of Phosphatidylserine in different concentrations of Thrombin with out Aspirin.
The first graphs is indicating that , adding Thrombin on its own could rise up externalising phosphatidylserine on the outer membrane of the platelets. This is while Aspirin had not been added to the samples and Alcohol (ethanol) was used instead of Aspirin. As the chart suggests the general trend is increasing.
5.1.2. The next stage is to evaluate the role of Aspirin. The first eight tubes are with out Aspirin (Solution A had been added).The mean of fluorescent intensity for different concentration of Thrombin would be me calculated. For tubes from 8 to 16, solution B had been added which was including Aspirin. Then the mean of Fluorescent intensity for different concentration of Thrombin for these tubes would be taken as well. They should be compared with each other. The result comes below.
Chart 5.1.2.1. Compare the effect of Aspirin on different concentrations of Thrombin.
The concentration of each tube has been mentioned. The added Aspirin per each tube was 2µL. As the graph suggests, in most cases the ADP causes decreasing in externalisation of Thrombin although the change is not significant( P<0.05) however in this regard in a research conducted by Conehn Zoë and colleagues in 2004, revealed that when the plateletes become inhibited they express less phosphatidylserine on the outer membrane. This matches with the achievements of our experiment.
5.2. ADP and Aspirin
Before doing this experiment another method was used in order to get proper data for ADP. But as results were not satisfactory therefore only the double amount of final concentration of ADP is used in new method. In previous method the range of concentration of ADP was similar to Thrombin in current method and highest concentration of ADP was 10µL while in current method the concentration of ADP is 20 µL. Perhaps running this method will not reach a visible result about ADP. The role of ADP in activating the platelets has already been proved (Jianguo Jin et al, 1998) .The fact that activating by ADP cause measurable change is not something which could be found by this experiment. Maybe by applying different methods, better results are reached. Therefore the first chart (like Thrombin) will be excluded.
5.2.1. This stage is to evaluate the effect of Aspirin on platelets which have already been activated by ADP. The tube 8 is include 20µL ADP with out Aspirin (Solution A had been added) and tube 16 is include 20µL ADP with Aspirin. In five times of running the experiment the mean of fluorescent intensity of tubes 8 and 16 would be me calculated and the results are compared with each other. The concentration of added Aspirin as mentioned in method is 0.01 % V/V. The important factor in this regard is the amount of Aspirin used for each tube.
Chart 5.2.2.1. Aspirin has effect on platelets which had already been activated by ADP.
By this chart it is not quite clear to recognise what is the effect of Aspirin of the specimen which have already been activated with ADP. May be there are some interactions between the function of ADP and Aspirin in platelets cell membrane which can cause a visible change. But apart from specimen 4 which has a high SD (as the Y-error bars shows); generally it is logic to say that Aspirin cause internalisation the phosphatidylserine. We should not forget that we are looking for effect of Aspirin (either internalising or externalising). If platelets have already been activated with different activators although they may cause overlap, but the most important impact should not be forgotten. The next chart will reveals more valuable information in this regard.
Additionally, as stated before Aspirin Induces Apoptosis through -Release of Cytochrome c from Mitochondria (Katja C Zimmermann et al, 2000) and the inhibition of Proteasome Function (Priyanka Dikshit et al, 2006). It is logic and true. When a cell is inhibited for along time and is not in use, the final destination is not something apart from apoptosis. Although the main cause of apoptosis is not what was mentioned, but inhibition a cell.
5.3. Control and Aspirin
In order to prove the hypothesis, the sole effect of Aspirin on platelets which have not been activated by any factor would be evaluated as well. This is a compare between controls of samples which have been added Alcohol (from solution A) and the control of samples which have been added Aspirin (from solution B). Chart comes below manifest this compare.
Chart 5.2.2.1. Compare Controls with and with out Aspirin.
Here a significant change is observed (P<0.01). href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Casta%C3%B1o%20E%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract">Castaño E and colleagues in 1999 announced that Aspirin increases phosphatidylserine externalization when they were analysing HT-29 colon carcinoma cells .In this paper is said that Aspirin induces cell death and caspase-dependent phosphatidylserine externalization. Based on their paper one of two halls marks of apoptosis is: increase in phosphatidylserine externalization. : It does not correspond with other findings which discuss about inhibitory role of Aspirin. As mentioned before, when platelets become activated they express more phosphatidylserine on the outer leaflet, therefore when they become inhibited (for example by Aspirin) they logically must express less phosphatidylserine while bases on the aforementioned paper was not. But Pamela L in her book demonstrate that unlike observation about colorectal cell lines , if the method of measuring is based on Annexin V binding to phosphatidylserine, there was not dose dependent apoptotic increase .
5.1. Thrombin and Aspirin
5.1.1. The first chart will demonstrate the externalisation of the Thrombin in different concentrations. It is with out Aspirin .This chart would reveal that what is the effect of Thrombin in externalisation of Phosphatidylserine with out Aspirin.
Graph 5.1.1.1. Externalising of Phosphatidylserine in different concentrations of Thrombin with out Aspirin.
The first graphs is indicating that , adding Thrombin on its own could rise up externalising phosphatidylserine on the outer membrane of the platelets. This is while Aspirin had not been added to the samples and Alcohol (ethanol) was used instead of Aspirin. As the chart suggests the general trend is increasing.
5.1.2. The next stage is to evaluate the role of Aspirin. The first eight tubes are with out Aspirin (Solution A had been added).The mean of fluorescent intensity for different concentration of Thrombin would be me calculated. For tubes from 8 to 16, solution B had been added which was including Aspirin. Then the mean of Fluorescent intensity for different concentration of Thrombin for these tubes would be taken as well. They should be compared with each other. The result comes below.
Chart 5.1.2.1. Compare the effect of Aspirin on different concentrations of Thrombin.
The concentration of each tube has been mentioned. The added Aspirin per each tube was 2µL. As the graph suggests, in most cases the ADP causes decreasing in externalisation of Thrombin although the change is not significant( P<0.05) however in this regard in a research conducted by Conehn Zoë and colleagues in 2004, revealed that when the plateletes become inhibited they express less phosphatidylserine on the outer membrane. This matches with the achievements of our experiment.
5.2. ADP and Aspirin
Before doing this experiment another method was used in order to get proper data for ADP. But as results were not satisfactory therefore only the double amount of final concentration of ADP is used in new method. In previous method the range of concentration of ADP was similar to Thrombin in current method and highest concentration of ADP was 10µL while in current method the concentration of ADP is 20 µL. Perhaps running this method will not reach a visible result about ADP. The role of ADP in activating the platelets has already been proved (Jianguo Jin et al, 1998) .The fact that activating by ADP cause measurable change is not something which could be found by this experiment. Maybe by applying different methods, better results are reached. Therefore the first chart (like Thrombin) will be excluded.
5.2.1. This stage is to evaluate the effect of Aspirin on platelets which have already been activated by ADP. The tube 8 is include 20µL ADP with out Aspirin (Solution A had been added) and tube 16 is include 20µL ADP with Aspirin. In five times of running the experiment the mean of fluorescent intensity of tubes 8 and 16 would be me calculated and the results are compared with each other. The concentration of added Aspirin as mentioned in method is 0.01 % V/V. The important factor in this regard is the amount of Aspirin used for each tube.
Chart 5.2.2.1. Aspirin has effect on platelets which had already been activated by ADP.
By this chart it is not quite clear to recognise what is the effect of Aspirin of the specimen which have already been activated with ADP. May be there are some interactions between the function of ADP and Aspirin in platelets cell membrane which can cause a visible change. But apart from specimen 4 which has a high SD (as the Y-error bars shows); generally it is logic to say that Aspirin cause internalisation the phosphatidylserine. We should not forget that we are looking for effect of Aspirin (either internalising or externalising). If platelets have already been activated with different activators although they may cause overlap, but the most important impact should not be forgotten. The next chart will reveals more valuable information in this regard.
Additionally, as stated before Aspirin Induces Apoptosis through -Release of Cytochrome c from Mitochondria (Katja C Zimmermann et al, 2000) and the inhibition of Proteasome Function (Priyanka Dikshit et al, 2006). It is logic and true. When a cell is inhibited for along time and is not in use, the final destination is not something apart from apoptosis. Although the main cause of apoptosis is not what was mentioned, but inhibition a cell.
5.3. Control and Aspirin
In order to prove the hypothesis, the sole effect of Aspirin on platelets which have not been activated by any factor would be evaluated as well. This is a compare between controls of samples which have been added Alcohol (from solution A) and the control of samples which have been added Aspirin (from solution B). Chart comes below manifest this compare.
Chart 5.2.2.1. Compare Controls with and with out Aspirin.
Here a significant change is observed (P<0.01). href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Casta%C3%B1o%20E%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract">Castaño E and colleagues in 1999 announced that Aspirin increases phosphatidylserine externalization when they were analysing HT-29 colon carcinoma cells .In this paper is said that Aspirin induces cell death and caspase-dependent phosphatidylserine externalization. Based on their paper one of two halls marks of apoptosis is: increase in phosphatidylserine externalization. : It does not correspond with other findings which discuss about inhibitory role of Aspirin. As mentioned before, when platelets become activated they express more phosphatidylserine on the outer leaflet, therefore when they become inhibited (for example by Aspirin) they logically must express less phosphatidylserine while bases on the aforementioned paper was not. But Pamela L in her book demonstrate that unlike observation about colorectal cell lines , if the method of measuring is based on Annexin V binding to phosphatidylserine, there was not dose dependent apoptotic increase .
4. Experimental results
The results of flowcytometry of each tube come below. The experiment was performed for five times. Hence it is expected to see the five series of sixteen results.
In each group of results two diagrams and one table are seen. Details of each result come below (Guide line of the flowcytometry machine).
-Upper diagram
FSC Forward Scatter: Cell size
SSC Side Scatter: Granularity
-Lower diagram
X scatter: Fluorescent intensity emitted is proportional to the quantity of binding sites for the fluorescent compound on the cell/particle. The unit is MESF which is Molecules of Equivalent Soluble Fluorochrome. In this experiment the geometric mean of fluorescent is measured.
Y scatter: Events count
-Table
Geometric mean of all events
Geometric mean of M1 region (1% of the events)
FL2-H: 585/42 nm, yellow-green colour
Mean: Average X-axis channel number of linear value for events in the quadrant
Geometric Mean: average of the logarithm of the X-axis channel number or linear value for events in the quadrant expressed anti-log.
Picture 4.1.1. Flowcytometry machine.
BD FCSCalibur two laser channel.
4.1. Diagrams and table from flowcytometry.
Results of flowcytometry for each sample come next.
The results of flowcytometry of each tube come below. The experiment was performed for five times. Hence it is expected to see the five series of sixteen results.
In each group of results two diagrams and one table are seen. Details of each result come below (Guide line of the flowcytometry machine).
-Upper diagram
FSC Forward Scatter: Cell size
SSC Side Scatter: Granularity
-Lower diagram
X scatter: Fluorescent intensity emitted is proportional to the quantity of binding sites for the fluorescent compound on the cell/particle. The unit is MESF which is Molecules of Equivalent Soluble Fluorochrome. In this experiment the geometric mean of fluorescent is measured.
Y scatter: Events count
-Table
Geometric mean of all events
Geometric mean of M1 region (1% of the events)
FL2-H: 585/42 nm, yellow-green colour
Mean: Average X-axis channel number of linear value for events in the quadrant
Geometric Mean: average of the logarithm of the X-axis channel number or linear value for events in the quadrant expressed anti-log.
Picture 4.1.1. Flowcytometry machine.
BD FCSCalibur two laser channel.
4.1. Diagrams and table from flowcytometry.
Results of flowcytometry for each sample come next.
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.
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.
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