This protocol allows us to answer a variety of questions about the way that shear acts on cells in solution and provides significant control over shear duration and magnitude. Unlike other techniques, which detect the effect of shear on cells that are immobilized, this technique allows us to apply shear to cells in solution and lends itself to a spectrum of downstream signal detection methods. After obtaining peripheral blood from a consenting, healthy adult donor in a 4.5-milliliter tube of 3.8%trisodium citrate, centrifuge the blood to obtain platelet-rich plasma, and use a pipette tip cut at a 45-degree angle to carefully remove the resulting top, cloudy, yellow platelet-rich plasma layer.
Obtain the platelet count via complete blood count, and adjust the platelet count to approximately 2.5 times 10 to the fifth platelets per microliter with pooled human platelet-poor plasma. Then, place the suspension at 22 degrees Celsius with gentle rotation. 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 five 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 two 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 five 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. Platelet activation marker expression in response to shear force application is unregulated in a time-dependent manner. Here, representative readouts of human platelet activation in the presence of two antibodies targeting the N-terminal domain of glycoprotein 1b-IX and one control antibody under sheared and static conditions are shown.
The expression of all three activation markers was significantly increased in the presence of shear forces and targeting antibodies, compared to either static conditions or in the presence of control antibodies. Notably, treatment with antibody with an unbinding force too low to trigger glycoprotein 1b-IX activation does not trigger platelet activation marker upregulation under static or shear force conditions. Further, plasma from immune thrombocytopenia patients containing antibodies against glycoprotein 1b-IX trigger shear dependent responses in contrast with plasma from patients without glycoprotein 1b-IX targeting antibodies.
In addition of flowcytometric analyses, cells that have been exposed to shear can also be lysed for western blot, ELISA, and mass-spec analysis to determine any changes in protein level. As always, when using blood or human samples, be sure to wear proper protective equipment and follow the lab safety and blood born pathogen guidelines of your institution.
