Read about the following methods and write about them in details....
Question
Read about the following methods and write about them in details.
Project methods
1- Washed platelet preparation
To prepare the platelet-rich plasma, anticoagulant blood will be centrifuged at 250g for 15 min. The PRP will be drawn off and transferred to a conical 50 ml centrifuge tube and 1 µg/ml prostacyclin (PGI2) will be added to prevent the platelets becoming activated. The sample will be centrifuged at 1000g for 10 min. The resultant platelet pellet will be suspended in 3 ml of ACD, with 25 ml Tyrode’s buffer (134 mM NaCl, 2.9 mM KCl, 1 mM MgCl2, 20 mM HEPES, 12mM NaHCO3, 5 mM glucose) (pH 7.3). A further 1 µg/ml PGI2 will be added to the sample and then centrifuged again at 1000g for 10 min. Finally, the platelets will be re-suspended in Tyrode’s buffer.
2- Flow Cytometry
Flow cytometry was performed to detect the activation markers on platelet surface including P-selectin expression and PAC1 binding. Washed platelets treated with various concentrations of HbS or platelets isolated from patients and controls were labeled with anti-P-selection FITC or PAC1 FITC antibody. The platelet surface expression of above proteins was measured by flow cytometry (Becton Dickinson, San Jose, CA, USA) and expressed as the mean fluorescence.
3- Confocal fluorescent microscopy
Anti-P selection FITC antibody was added to washed human platelets (0.5 × 106/mL) and smeared on glass cover slides pre-coated with BSA or HbS, and incubated for 30–45 min at 37°C. The BSA (2%) or HbS (1.5 μM) was incubated on cover slides for 1 hr at 37°C for immobilization. The HbS coated surface was further incubated with 2% BSA for 30 min at RT. After gentle wash with modified Tyrode buffer, slides with adhered-platelets were mounted with Fluoromount-G (Southern Biotech Assoc., Birmingham, AL, USA) and image was captured using a confocal microscope.
4- Measurement of extracellular Hb level in plasma
The Hb concentration in plasma was measured using the sandwich ELISA. Briefly, plasma (100 μL of 1:5,000 dilutions) from patients or normal individuals was incubated in microtiter plates coated with rabbit anti-Hb antibody (Sigma-Aldrich, St. Louis, USA) for 1 hr at RT. The bound Hb was detected by an affinity purified goat anti-Hb conjugated with HRP (Abcam, Cambridge, USA) using TMB substrate at an OD ~ 450 nm. Plasma Hb concentration was calculated as μM compared to human erythrocyte purified HbS (Sigma-Aldrich, St. Louis, USA).
5- Microparticles measurement from plasma
Plasma microparticles (MPs) were isolated from 5 mL of citrate anti-coagulated plasma. Platelet-free plasma (PFP) was obtained by 3 sequential centrifugations of blood at 700 g for 6 min; platelet-rich plasma at 1500 g for 7 min; and platelet-poor plasma at 1500 g for 15 min. Platelet-derived MPs were measured from PFP in filtered-PBS using anti-CD41 PE antibody and quantified using flow cytometry.
6-Platelet aggregation
Turbidimetry, which represents platelet aggregation, will be measured using a dual channel lumi-aggregometer. To set the scale before aggregation is assessed, PRP and PPP will be used. Samples (396 μl) will be pre-warmed for 120 s at 37° C then stirred at 1200 rpm; an ADP agonist will be added at a dilution of 1:100. Platelet aggregation will be determined by comparing the optical density of the sample against that of the PPP control. Platelet aggregation will be monitored for 5 min.
7- Coagulation assay
Prothrombin time (PT) and the activated partial thromboplastin time (APTT) will be measured using STAGO Start4 instrument. The steel balls will be added to the cuvettes before adding them to the wells of instrument. For PT assay, a 50 µL of patient plasma will be incubated for 3 minutes at 37C and clotting will be initiated by adding 100µL of STA-Neoplastine Cl plus reagent. The result will be recorded by analyzer. For APTT essay, 50 µL of patient plasma with 50 µL of STA-PTT reagent will be incubated for 3 minutes at 37C. Clotting will be initiated by adding 50 µL of CaCl2.
8- Latex-based VWF
This test will use for determination of the ristocetin cofactor activity of von Willebrand factor in human plasma using the platelet agglutination method. 50 µL plasma dilution or blank will be mixed with 50 µL of Reagent followed by agitation slowly for 1 minute. After 1-minute agglutination reaction will be observed. Semiquantitative test will be performed on diluted plasma sample. A positive plasma sample will be serially diluted twofold with normal saline. The highest dilution that still shows a positive reaction is the end-titre of a sample.
Project methods
1- Washed platelet preparation
To prepare the platelet-rich plasma, anticoagulant blood will be centrifuged at 250g for 15 min. The PRP will be drawn off and transferred to a conical 50 ml centrifuge tube and 1 µg/ml prostacyclin (PGI2) will be added to prevent the platelets becoming activated. The sample will be centrifuged at 1000g for 10 min. The resultant platelet pellet will be suspended in 3 ml of ACD, with 25 ml Tyrode’s buffer (134 mM NaCl, 2.9 mM KCl, 1 mM MgCl2, 20 mM HEPES, 12mM NaHCO3, 5 mM glucose) (pH 7.3). A further 1 µg/ml PGI2 will be added to the sample and then centrifuged again at 1000g for 10 min. Finally, the platelets will be re-suspended in Tyrode’s buffer.
2- Flow Cytometry
Flow cytometry was performed to detect the activation markers on platelet surface including P-selectin expression and PAC1 binding. Washed platelets treated with various concentrations of HbS or platelets isolated from patients and controls were labeled with anti-P-selection FITC or PAC1 FITC antibody. The platelet surface expression of above proteins was measured by flow cytometry (Becton Dickinson, San Jose, CA, USA) and expressed as the mean fluorescence.
3- Confocal fluorescent microscopy
Anti-P selection FITC antibody was added to washed human platelets (0.5 × 106/mL) and smeared on glass cover slides pre-coated with BSA or HbS, and incubated for 30–45 min at 37°C. The BSA (2%) or HbS (1.5 μM) was incubated on cover slides for 1 hr at 37°C for immobilization. The HbS coated surface was further incubated with 2% BSA for 30 min at RT. After gentle wash with modified Tyrode buffer, slides with adhered-platelets were mounted with Fluoromount-G (Southern Biotech Assoc., Birmingham, AL, USA) and image was captured using a confocal microscope.
4- Measurement of extracellular Hb level in plasma
The Hb concentration in plasma was measured using the sandwich ELISA. Briefly, plasma (100 μL of 1:5,000 dilutions) from patients or normal individuals was incubated in microtiter plates coated with rabbit anti-Hb antibody (Sigma-Aldrich, St. Louis, USA) for 1 hr at RT. The bound Hb was detected by an affinity purified goat anti-Hb conjugated with HRP (Abcam, Cambridge, USA) using TMB substrate at an OD ~ 450 nm. Plasma Hb concentration was calculated as μM compared to human erythrocyte purified HbS (Sigma-Aldrich, St. Louis, USA).
5- Microparticles measurement from plasma
Plasma microparticles (MPs) were isolated from 5 mL of citrate anti-coagulated plasma. Platelet-free plasma (PFP) was obtained by 3 sequential centrifugations of blood at 700 g for 6 min; platelet-rich plasma at 1500 g for 7 min; and platelet-poor plasma at 1500 g for 15 min. Platelet-derived MPs were measured from PFP in filtered-PBS using anti-CD41 PE antibody and quantified using flow cytometry.
6-Platelet aggregation
Turbidimetry, which represents platelet aggregation, will be measured using a dual channel lumi-aggregometer. To set the scale before aggregation is assessed, PRP and PPP will be used. Samples (396 μl) will be pre-warmed for 120 s at 37° C then stirred at 1200 rpm; an ADP agonist will be added at a dilution of 1:100. Platelet aggregation will be determined by comparing the optical density of the sample against that of the PPP control. Platelet aggregation will be monitored for 5 min.
7- Coagulation assay
Prothrombin time (PT) and the activated partial thromboplastin time (APTT) will be measured using STAGO Start4 instrument. The steel balls will be added to the cuvettes before adding them to the wells of instrument. For PT assay, a 50 µL of patient plasma will be incubated for 3 minutes at 37C and clotting will be initiated by adding 100µL of STA-Neoplastine Cl plus reagent. The result will be recorded by analyzer. For APTT essay, 50 µL of patient plasma with 50 µL of STA-PTT reagent will be incubated for 3 minutes at 37C. Clotting will be initiated by adding 50 µL of CaCl2.
8- Latex-based VWF
This test will use for determination of the ristocetin cofactor activity of von Willebrand factor in human plasma using the platelet agglutination method. 50 µL plasma dilution or blank will be mixed with 50 µL of Reagent followed by agitation slowly for 1 minute. After 1-minute agglutination reaction will be observed. Semiquantitative test will be performed on diluted plasma sample. A positive plasma sample will be serially diluted twofold with normal saline. The highest dilution that still shows a positive reaction is the end-titre of a sample.
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1- Washed platelet preparation
Tyrode’s buffer (134 mMNaCl, 2.9 mMKCl, 1 mM MgCl2, 20 mM HEPES, 12mM NaHCO3, 5 mM glucose) (pH 7.3) Keep the buffer prepared. Depending on experiment, warm buffer up to room temperature or keep on ice before use.
ACD buffer (acid-citrate-dextrose)
39 mM citric acid, 75 mM sodium citrate, 135 mM dextrose, pH 7.4. Before use, warm up to room temperature and adjust pH if necessary
Centrifuge result :
Bottom layer: Red blood cells (accounting for 50–80% of the total volume)
Middle layer: Very thin band of white blood cells (also called "buffy coat")
Top layer: Straw-colored PRP
1. Take anticoagulant added blood sample
2. Centrifuge at 250g for 15 min
3. Take the plasma rich portion (PRP) and put it in to a conical 50 ml centrifuge tube
4. Add 1 µg/ml prostacyclin (PGI2)—this will prevent activation
5. Centrifuge again for 10min at 1000g
6. Take the pellet
7. Put the pellet in 3 ml of ACD, with 25 ml Tyrode’s buffer
8. Again 1 µg/ml PGI2 is added to the sample and then centrifuged again at 1000g for 10 min
9. platelets will be re-suspended in Tyrode’s buffer
2 - FLOW CYTOMETRY
Flow cytometry is a widely used method for analyzing the expression of cell surface and intracellular molecules, characterizing and defining different cell types in a heterogeneous cell population, assessing the purity of isolated subpopulations and analyzing cell size and volume. It allows simultaneous multi-parameter analysis of single cells.
It is predominantly used to measure fluorescence intensity produced by fluorescent-labeled antibodies detecting proteins, or ligands that bind to specific cell-associated molecules such as propidium iodide binding
Tyrode’s buffer (134 mMNaCl, 2.9 mMKCl, 1 mM MgCl2, 20 mM HEPES, 12mM NaHCO3, 5 mM glucose) (pH 7.3) Keep the buffer prepared. Depending on experiment, warm buffer up to room temperature or keep on ice before use.
ACD buffer (acid-citrate-dextrose)
39 mM citric acid, 75 mM sodium citrate, 135 mM dextrose, pH 7.4. Before use, warm up to room temperature and adjust pH if necessary
Centrifuge result :
Bottom layer: Red blood cells (accounting for 50–80% of the total volume)
Middle layer: Very thin band of white blood cells (also called "buffy coat")
Top layer: Straw-colored PRP
1. Take anticoagulant added blood sample
2. Centrifuge at 250g for 15 min
3. Take the plasma rich portion (PRP) and put it in to a conical 50 ml centrifuge tube
4. Add 1 µg/ml prostacyclin (PGI2)—this will prevent activation
5. Centrifuge again for 10min at 1000g
6. Take the pellet
7. Put the pellet in 3 ml of ACD, with 25 ml Tyrode’s buffer
8. Again 1 µg/ml PGI2 is added to the sample and then centrifuged again at 1000g for 10 min
9. platelets will be re-suspended in Tyrode’s buffer
2 - FLOW CYTOMETRY
Flow cytometry is a widely used method for analyzing the expression of cell surface and intracellular molecules, characterizing and defining different cell types in a heterogeneous cell population, assessing the purity of isolated subpopulations and analyzing cell size and volume. It allows simultaneous multi-parameter analysis of single cells.
It is predominantly used to measure fluorescence intensity produced by fluorescent-labeled antibodies detecting proteins, or ligands that bind to specific cell-associated molecules such as propidium iodide binding
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