To begin, prepare the appropriate coating solution in modified Tyrode's buffer and coat an eight-well microslide by incubating it for two hours at 37 degrees Celsius. Then wash the coated microslide twice with modified Tyrode's buffer. Incubate the microslide in modified Tyrode's buffer containing five milligrams per milliliter BSA for at least 30 minutes to quench nonspecific adhesion.
To prepare washed platelets, centrifuge citrate anticoagulated whole blood at 200 G for 20 minutes to obtain platelet-rich plasma. Then add indomethacin and PGE1 to the platelet-rich plasma and centrifuge at 500 G for 10 minutes. Resuspend the resulting platelet pellet in modified Tyrode's HEPES buffer at 37 degrees Celsius to achieve a density of four times 10 to the power of seven platelets per milliliter.
Allow the isolated platelets to rest for 30 minutes at 37 degrees Celsius. Next, add DHE solution to the platelet suspension to achieve a final concentration of 10 micromolar and incubate at 37 degrees Celsius for one minute. After incubation, remove the blocking solution from the microslide and position it on the microscope stage for imaging.
Then gently dispense the platelets. Use a 40X oil immersion lens on an inverted confocal microscope to monitor the conversion of DHE to 2-hydroxyethidium. Image for 10 minutes, collecting images every 10 seconds.
To monitor superoxide anion generation in response to an agonist, add a soluble agonist 10 minutes after dispensing the platelets and collect fluorescence images for an additional 10 minutes. Using this protocol, the generation of superoxide radicals was studied during platelet adhesion to collagen or fibrinogen, and from platelets adhered to PLL stimulated with thrombin.
