Use of the modified parallel plate flow chamber can help researchers answer key questions pertaining to the functional response of mechanosensitive ion channels to shear stress. The main advantage of this technique is that it allows real-time investigation to flow activated ion channels in an easily assembled, reusable parallel plate flow chamber. To begin the procedure, apply a thin layer of silicone elastomer solution around the edges of the rectangular space of piece C, and gently place the rectangular cover glass, piece D, directly on the elastomer solution to completely cover the open rectangular space of piece C.Carefully wipe away excess silicone elastomer solution.
Then, repeat the procedure for adhering the rectangular cover glass, piece F, to the bottom of piece E and allow the silicone elastomer solution to cure overnight at room temperature. Now, assemble the MPP flow chamber by sequentially placing each piece on top of the previous, beginning with the bottom chamber, piece E, then piece C, piece B, followed by piece A on the top. Next, align the screw holes of each piece at the corners and tightly screw the pieces together to prevent leaks from occurring while administering flow to the MPP flow chamber.
In a six-well plate, place four to five, 12 millimeter cover glass circles in each well. Save the cells between 10%and 30%confluency, such that single cells can be accessed for electrophysiological recordings. Subsequently, incubate the cells under standard culture conditions for no less than two hours to allow cells to adhere, and no more than 24 hours, as endothelial cells will become flat and difficult to patch when seeded at subconfluency for more than 24 hours.
Next, remove a cover glass containing the adhered cells from a well of the six-well plate and quickly rinse in PBS. Then, transfer the cover glass with cells to a 35 millimeter Petri dish containing two milliliters of electrophysiological BAF solution. Immediately transfer the cover glass with cells to the MPP flow chamber.
Transfer the cover glass circle to the rectangular cover glass, piece D, which is adhered to piece C of the MPP flow chamber. Add the BAF solution to completely submerge the cover glass circle and the cells. Subsequently, position the cover glass circle on piece D such that the cells will be in line with the slit openings of piece B.Ensure that the cover glass circle adheres to piece D through solution glass adhesion to avoid disruption of the cover glass circle's position by the flow application.
Then, assemble the MPP flow chamber by screwing the pieces together in the appropriate order. Transfer the chamber to the microscope stage and immediately perfuse the chamber with BAF solution. Next, identify a healthy cell with a dark border and obvious nucleus for the experiment.
Avoid cells that appear to be blubbing or cells that are in contact with other cells. To control shear stress, set up a gravity perfusion system by connecting a 30 milliliter graduated syringe cylinder to a three-way Luer lock fitted with microbore tubing. Next, attach the graduated cylinder to the Faraday cage surrounding the electrophysiology rig using double-sided tape.
Prior to inserting the tubing in the MPP chamber, pre-fill the syringe and tubing with BAF solution. Then, insert the tubing into the MPP flow chamber inlet hole of piece A.Pre-fill the MPP flow chamber with solution, such that it is being removed in the vacuum reservoir. Stop the flow to the chamber and refill the graduated cylinder to the top mark.
Calculate the flow rate manually using a stopwatch by allowing the solution to flow through the chamber at a given syringe cylinder height. Raise or lower the syringe to alter the flow and calculate the shear stress in a parallel chamber using this equation. Repeat this process until a desired level of shear stress is found.
Subsequently, transfer an assembled chamber containing adhered cells to the microscope stage of the electrophysiology rig. And insert the tubing, pre-filled with BAF solution, into the hole of piece A.Then, fill the chamber and wash the cells with 10 milliliters of BAF solution, simultaneously. Once the desired patch configuration is successfully obtained, allow channel currents to stabilize in a static bath at room temperature.
As soon as the currents have stabilized, apply shear in a step-wise fashion, allowing the increased current to stabilize prior to the next step of shear stress increase. Remove shear exposure to the cells by stopping the flow to the chamber, allowing mechanosensitive channel currents to return to baseline currents observed in the static bath. Shown here is a representative raw recording of Kir current from a primary mouse mesenteric endothelial cell with notable linear outward leak current.
A ramp of negative 140 to positive 40 millivolts was applied to the patch over 400 milliseconds. Leak current prevents the analysis of real Kir channel activity. To subtract leak current, first calculate the linear slope conductance of leak current.
Multiple G slope by corresponding voltages of the entire raw trace to plot leak current on the raw data. The line should overlay the outward linear leak exactly. Then, subtract the plotted leak current from the entire trace so that the linear outward current is about zero picoamperes per picofarad, and the real Kir current can be analyzed.
The most important thing to remember when attempting this procedure is to properly position the cover glass in the chamber so that cells are accessible for experiments. In addition, be sure that the cover slip is adequately adhered to piece D, such that fluid flow does not disrupt the cover glass containing cells.
