The Effects of Shear Stress on Platelets Treated with Antibodies Against Mechanosensory Receptors
Transcript
For ligand treatment, slowly, add the antibody or ligand of interest to the platelet-rich plasma, and mix the platelets gently. During the incubation, turn on the cone-plate viscometer, and set the plate temperature to 22 degrees Celsius. After 5 to 10 minutes, transfer the treated platelet-rich plasma onto the temperature-controlled cone-plate viscometer directly at the center of the plate, taking care that all of the sample is deposited between the cone and plate at the point of contact.
To apply shear to the sample, calculate the shear according to the viscometer manual, and apply shear to the sample at the appropriate rate and duration. At the end of the application, lift the cone about 2 millimeters off the plate, so that the sample remains in contact with both the plate and the cone, and use a gel-loading pipette tip to collect 5 to 10 microliters from the center of the sample volume. Then, incubate the sheared sample with antibodies against the surface markers of interest for 20 minutes at room temperature, and fix the samples in 2% paraformaldehyde for 20 minutes at room temperature.
To analyze the samples by flow cytometry, collect at least 20,000 events for each condition, and quantify the signal strength of each fluorescent marker using the height value for the intensity of each fluorophore. Then, use a negative control with bovine serum albumin or vehicle to draw a gate excluding negative background fluorescent events, and quantify the percentage of events inside the gate for all experimental conditions.
