Begin the protocol by preparing the shear assay viscous flow media. To do so, preheat the base culture media for about 10 to 20 minutes at 60 to 70 degrees Celsius using a magnetic stir or hot plate. While continuously stirring the media, gently add methylcellulose into it to help the methylcellulose particles disperse quickly without coagulating.
Allow this process to continue for approximately 15 to 24 hours to ensure a clear homogenous mix of media and cellulose. To set up the shear apparatus, attach a rubber gasket to the flow path to fasten the connection between the flow chamber and the Petri dish, and ensure a controlled uniform flow of single cells. The rubber gasket comes in different sizes depending on the desired flow profile and area of observation.
Next, set up the shear assay system comprising of 60 or 100 milliliter dual syringes connected to a programmable syringe pump for infusion and withdrawal of the viscous culture media. Fill a syringe with the prepared viscous flow media and attach the filled syringe to its respective location on the syringe pump. Then, attach an empty syringe to the other syringe location on the syringe pump.
Using 1/16th inch tubing and tubing connectors, connect both syringes to the flow chamber. Program the pump to infuse and withdraw a certain volume of fluid at a designated rate, and select the corresponding syringes. An example program is shown on the screen.
Aspirate the cell culture media from the Petri dish to prepare for shear. Next, insert and fix the flow chamber with the rubber gasket onto the Petri dish containing the attached cells. Place the fitted microfluidic flow chamber with the cells in the dish onto an inverted microscope connected to a display monitor.
The setup is now ready for applying fluid shear stress onto a single cell and optically monitoring the resulting cellular deformation.
