Biology
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Summary May 7th, 2019
Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.
Transcript
Platelets should be free from blood cells and proteins when studying their biochemical and physiological properties. Therefore, we have developed a protocol to purify platelets from mouse blood. This technique can purify platelets using iohexol gradient medium and low-speed centrifugation, resulting in a clear separation of platelets from other blood components.
To purify the platelets, use a wide-bore pipette tip to layer 200 microliters of freshly harvested whole blood slowly down the side of a 1.5-milliliter tube onto 600 microliters of iohexol gradient medium without mixing. After centrifugation in a swinging-bucket rotor to isolate the platelets, use a new wide-bore pipette tip to collect most of the platelet-rich layer and a small fraction of the platelet-poor layer without aspirating the white blood cell or red blood cell layer. Add the platelet sample to a new tube, and add one milliliter of PBS to the platelets.
Mix the platelets by inversion before performing another centrifugation. Then resuspend the pellet in 200 microliters of PBS with mild pipetting. For platelet activation, transfer one to two times 10 to the six platelets to a new tube containing 100 microliters of staining buffer.
Add one to two times 10 to the six platelets to a different tube containing 100 microliters of staining buffer supplemented with 0.4-millimolar GPRP peptide. Add thrombin to the tube with the GPRP peptide to activate the platelets, and add the antibody cocktail of interest to both tubes. As a positive control, add one microliter of whole blood in up to 100 microliters of staining buffer in a tube containing 0.4-millimolar GPRP peptide, and add thrombin to activate these platelets.
As a negative control, add one microliter of whole blood in up to 100 microliters of staining buffer. Then stain both control tubes with the antibody cocktail. After 30 minutes at room temperature in the dark, wash the samples with one milliliter of fresh staining buffer per tube.
Resuspend the pellets in 300 microliters of fresh staining buffer. Then transfer the cell samples into individual flow cytometry analysis tubes protected from light. To analyze the platelets by flow cytometry, create a new experiment, and generate logarithmic forward scatter versus logarithmic side scatter dot plots.
After adjusting the voltages as necessary, record the single-color-stained cells for the compensation controls. Now run the positive control sample to acquire enough cells to adjust the compensation and the gates. Then acquire and record 100, 000 events for each whole blood sample, and analyze the flow cytometry data using the appropriate plots and gates.
If the blood is added onto the medium slowly, the iohexol gradient medium and the blood sample will form two separate layers in the tube. After centrifugation, the top, straw-colored layer contains the plasma but no intact blood cells. The second, whitish, platelet-rich layer contains the majority of the platelets.
The third, transparent layer is the platelet-poor layer and is directly above the red blood cell and white blood cell pellet. Whole blood exhibits distinct populations of red blood cells, white blood cells, and platelets on a logarithmic forward scatter versus side scatter dot plot. Purified platelets, however, demonstrate a distinct population with negligible numbers of red or white blood cells.
Whole blood samples contain Ter119-positive red blood cell and CD45-positive white blood cell populations that are nearly absent in purified platelet samples. Untreated purified platelets express almost no activation markers confirming their resting state, whereas purified thrombin-treated platelets typically demonstrate a 71%P-selectin expression. Microscopic evaluation of untreated purified platelet samples reveals no platelet aggregation.
After activation with thrombin, however, the platelets demonstrate a robust aggregation, further confirming their viability after purification as demonstrated. Layer the blood sample carefully onto the iohexol medium, and when collecting the platelets, be sure to aspirate carefully as to not collect the red blood cell and white blood cell layers for successful platelet purification. This method can be used for platelet purification from other species as well.
The purified platelets can be used for gene expression, activation, aggregation, granule release, and adhesion studies.
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