The overall goal of this experiment is to track platelet degranulation and secretion under flow using live-cell imaging. This method can help to answer key questions in the field of platelet biology. The main advantage of this technique is that it enables a versatile investigation of platelet contents at the subcellular level in live cells.
Though this method can provide insight into platelet function, it can also be applied to other cell types such as mast cells. In general, individuals new to this method, will struggle because of problems with platelet isolation in a form of preactivation. We first had the idea for this method when we realized that platelet secretion could only be evaluated at end point stages.
Real time images provide information of the release kinetics of platelet granular release. The procedure of platelet isolation will be demonstrated by Silvia Pignatelli, a student in our institute. Next, Arjan Barendrecht will demonstrate the real-time imaging experiments.
To begin, degrease cover glasses by incubating them overnight in undiluted chromosulfuric acid. The next day, rinse them with distilled water. Then place then overnight at 60 degrees Celsius in an oven, to dry.
Next, place a drop of water on a laboratory bench and adhere a 10 centimeter by 15 centimeter strip of a paraffin film on top of the drop by removing the air between the bench and the film. Then pipette 80 microliter drops of 10 microgram per milliliter von Willebrand factor or 100 microgram per milliliter fibrinogen onto the paraffin film. Place the dry cover glasses onto the drops and incubate them for one and a half hours at room temperature.
Next, add three milliliters of blocking buffer into each well of a four well plate. Transfer the cover glasses with the coated side up into individual wells of the plate. Incubate them overnight at four degrees Celsius.
Centrifuge nine milliliters of citrated whole blood for 15 minutes at 160 time G without a break. Then use a plastic Pasteur pipette to collect the supernatant containing the platelet-rich plasma. Add a one part of acid citrate dextrose to nine parts of the platelets by volume to prevent platelet preactivation.
Then centrifuge the platelets for 15 minutes at 400 times G without a break. Next, remove the supernatant, leaving a platelet pellet. Then resuspend the pellet carefully in HEPES Tyrode buffer at a pH of 6.5 back to the original volume of the platelet sample.
Then, add Prostacyclin to the resuspended platelets. Immediately centrifuge the sample and then remove the supernatant. Resuspend the pellet carefully in HEPES Tyrode buffer at a pH of 7.4.
Next, place the washed platelets into a cell analyzer and count the number of platelets. Adjust the number of platelets in the sample to a concentration of 150, 000 platelets per microliter with HEPES Tyrode buffer at a pH of 7.4. After resuspension, leave the platelets to recover for 30 minutes at room temp prior to the experiment.
Cover the top of the flow chamber with distilled water and place a customized silicone sheet with the smooth side down on the water. Float the sheet into position by removing excess water while keeping the sheet in position. Incubate the flow chamber for one hour at 60 degrees Celsius to evaporate the residual water and to firmly adhere the silicone sheet to the flow chamber.
With the plastic Pasteur pipette, add a drop of HEPES Tyrode buffer at a pH of 7.4 onto the flow channel. Then place the prepared cover glass with the coated side down, onto the flow channel. Turn on the vacuum pump to a pressure of around 10 kilopascals and attach the vacuum pump to the vacuum connection of the flow chamber.
Add 10 micrograms per milliliter DAPI stain to the washed platelets, and wrap the tube containing the platelets in aluminum foil. Then incubate them for 30 minutes at 37 degrees Celsius. To prevent platelet activation, add prostacyclin at a final concentration of 10 nanograms per milliliter and immediately centrifuge the platelets for 15 minutes at 400 times G without using the brake to remove unbound DAPI.
Reconstitute the platelet pellet and HEPES Tyrode buffer at a pH of 7.4 to the original volume with a concentration of 150, 000 platelets per microliter. Then, turn on the fluorescence microscope and load the microscope software. Set up the imaging parameters as described in table one of the accompanying text protocol.
Block the inlet tubing by connecting a 10 milliliter syringe and aspirating blocking buffer into the inlet tubing. Block for 15 minutes at room temperature and then rinse the inlet tubing by aspirating HEPES Tyrode buffer 7.4 into the inlet tubing. Connect the blocked inlet tubing and the untreated outlet tubing to the inlet and outlet of the chamber.
Then connect the 10 milliliter syringe to the outlet tubing. Place a drop of immersion oil on a 100 times objective and mount the chamber on the microscope with the glass surface facing downwards. Next, place the inlet tubing into a tube containing HEPES Tyrode buffer at a pH of 7.4.
Manually pull the plunger of the syringe connected to the outlet tubing to pull the solution through the chamber and remove the air from the system. Then place the syringe into the syringe pump and run the pump briefly to tighten the plunger and ensure stable flow. With the flow system now ready, add the antibodies and or dyes to the platelet suspension and mix carefully.
Then squeeze the inlet tubing and move it from the buffer to the tube containing the labeled platelets. Select live visualization in the software. Focus the microscope on the cover glass surface and start the pump at 484 microliters per minute.
Run the pump until the platelets reach the flow chamber. Next, reduce the flow rate to 7.5 microliters per minute. Monitor the cover glass surface.
Once the first platelet starts to adhere, focus the microscope on this platelet and start recording by initiating the experiment in the software. Record for 30 minutes and then stop the syringe pump. Save the microscope raw data files containing all the metadata.
When needed, export the movies to the MOV H 264 compressed or AVI uncompressed. Save separate frames as JPEG or TIF files. When finished imaging, remove the chamber from the microscope, clean the immersion oil from the glass, disconnect the vacuum and carefully remove the cover glass from the chamber with an 18 gauge needle.
Place the cover slide with the cells directed upwards in a new four well plate on top of a tissue prewetted with distilled water. Then pipette 750 microliters of a fixative solution on top of the cover glass and incubate it for one hour at room temperature in the dark. In case the samples cannot be analyzed directly, store them in 0.5%paraformaldehyde at four degrees Celsius to ensure stable fixation.
This shows the time series of platelet adhesion and spreading on glass slides coated with immobilized von Willebrand factor. Shown in green in CD63, a transmembrane protein which is mobilized onto the platelet surface in a time-dependent manner. Similarly, shown in orange, P-selectin is also a transmembrane protein inserted in the membrane of intracellular alpha-granules arresting platelets.
Following a flow experiment, confocal fluorescence microscopy can be used to produce a tilt series of the platelet adhesion. Seen here, CD63 is mainly located at the central granulomere while P-selectin is distributed over the entire cell body and appears concentrated at the edges. In these experiments, platelets, which do not have nuclear DNA, were preincubated with DAPI, which appears blue.
In this case, DAPI stains the polyphosphates and dense granules. Appearing later as green staining, is the time dependent mobilization of CD63 onto the platelet surface. Despite clear signs of dense granule release, single cell analysis shows that polyphosphate is retained within or on the outside of these platelets.
Once mastered, this technique can be done in six hours for four individual flow experiments. While attempting this procedure, it's important to prevent platelet preactivation. This can be achieved by careful manipulation of the cell suspension.
Following this procedure, other methods like confocal microscopy can be used to obtain additional insight into the three dimensional distribution of target molecules. After watching this video, you should have a good understanding of how to study platelet function under flow at a single cell level.
