The overall goal of this procedure is to observe and quantify von Willebrand factor or VWF-platelet string formation in response to secretagogues. This method can help answer key questions in Von Willebrand factor biology and wavo-palliative body pathophysiology. It also helps us answer questions about mutations that impair von Willebrand factor release and platelet binding function.
The main advantage of this technique is that it recapitulates the in-vivo interactions between sub-endothelial collagen, endoetholial cells VWF and platelets in a visual and quantifiable in-vitro system. I feel the visual demonstration of this method is vital. The required microscopy steps for capturing VWF-platelet string formation under multiple simultaneous conditions in real time can be difficult to master by text alone.
Begin by loading 0.5 milliliters of collagen coating buffer into a 1 milliliter luer lock syringe. Twist the end of the syringe onto the reservoir of a flow chamber slide and depress the plunger slowly until the entire lane is coated and each reservoir is full. Top up the reservoirs to ensure each one is full.
When all of the lanes have been loaded, incubate the chamber at 37 degrees Celsius for 24 hours. The next day, use a 200 microliter pipette to remove the collagen coating buffer. Then rinse each lane with HBSS, aspirating the saline with a 200 microliter pipette.
After the third round of HBSS has been discarded, add approximately three times 10 to the 6th Blood Outgrowth Endothelial Cells per milliliter in 100 microliters of growth medium, supplemented with 10%FBS to each lane of the flow chamber. Then return the chamber to the 37 degree Celsius incubator for 24 hours, changing the medium every eight to 12 hours. To assemble the flow apparatus, first attach one male elbow luer connector to one end of each of three 70 centimeter pieces of sterile silicone tubing.
And attach one female luer lock adapter to the other end of each tube. Connect the female luer lock adapters to individual 20 milliliter syringes and program the syringe pump. Load the syringes onto the pump, and tightly secure brackets around the syringes and plungers.
Then, attach individual male elbow luers to both ends of three different 20 centimeter pieces of sterile silicone tubing, and connect a blunted 18 gauge needle to one of each of the three elbow luers. Next, on a spinning disk microscope equipped with a fully automated stage, select the 20x objective and set the appropriate channel settings. To obtain the full dynamic data range, set the electron multiplying charge coupled device to an electron multiplying gain of 150 with an exposure time of 200 milliseconds.
Adjust the 491 nano-meter laser power to 20%and use the microscope software to mark the center of all three lanes. Set the Z-range to 1.0 micrometers and the step size to 0.5 micrometers. Finally, select the duration and the interval to capture an image every 10 seconds at 60 time points per slide.
To induce the formation of VWF-platelet strings replace the culture medium from three lanes of the flow chamber, with serum-free medium alone as a control, then the stimulant of interest diluted in 100 microliters of serum-free medium to the additional lanes. Then incubate the chamber for 15 minutes at room temperature. At the end of the stimulation period, wash all three of the lanes with 100 to 200 microliters of PBS, and use vascular clips to map the flow chamber onto a slide stand.
Attach one male luer connector from the end of a piece of 70 centimeter tubing to each reservoir of the flow chamber. And start the syringe pump to remove the PBS wash. Pipette additional PBS into the opposing flow chamber reservoir to ensure that the lanes do not run dry.
Once equivalent flow is confirmed, pause the syringe pump. Attach a 5 millileter syringe with a female luer lock coupler to the first free male luer connector. And submerge the blunted needle in a 1.5 milliliter micro-centrifuge tube containing flourescently labeled washed platelet solution.
Slowly, draw the platelet solution through the tubing to ensure that there are no air bubbles within the system. Attach the male luer to the open reservoir of the flow chamber, and load the other two lanes with platelet solution, as just demonstrated. Check the flow system setup to ensure all connections are secure.
Restart the syringe pump and image the platelet flow for 10 minutes in the dark. Then disconnect the luer from the intake tubing on the flow chamber reservoir. And fill the reservoir with PBS to maintain a gentle saline flow over the endothelial cell mono-layer.
To quantify the VWF-platelet string formation, capture 10 images per slide from each lane without overlap from the middle of the slide. When all of the images have been obtained, open the image processing software and click image, adjust, and brightness contrast. To adjust the brightness of the first image, dragging the scroll-bar until the adhered platelets can be viewed well enough for quantification.
Then, record a VWF-platelet string when three, or more, aligned platelets are observed and count all of the strings in the image. Following the initiation of flow conditions, the untreated cells demonstrate very few VWF-platelet strings compared to histamine, or PMA treated cells. Which exhibit an average of two to three strings per field of view.
Moreover, histones, which activate platelets as well as endothelial cells induce the production of more VWF-platelet strings than untreated, or histamine, or PMA stimulated controls. While attempting this procedure, keep the platelets quiescent until the flow experiment by discarding the first blood draw collection tube, maintaining all the reagents at room temperature and using a gentle pipette-ing technique. Modifications can be made to this procedure to evaluate VWF-platelet string formation in the presence of leukocytes, platelet activating agents, or string dissolution by the cleaving enzyme atom TS13.
After watching this video, you should have a good understanding of how to evaluate secretogogue-induced VWF-platelet string formation and in-vitro flow chamber model.
