Preparation of monolayers before mechanical strain

Preparation of MDCK monolayers before mechanical strain

Collagen coating of ISA or Live Cell Stretcher

• ISA or Live Cell Stretcher PDMS wells were loaded with 100-150ul of rat tail collagen solution in biosafety hood. Commercial collagen solutions can be substituted for the rat tail collagen.
• Collagen solution was left in wells for 30 minutes, then aspirated off.
• Devices were left in biosafety hood and sterilized with UV light for 45-60 minutes

Calcium Switch Plating of MDCK cells

• Sparsely plated and cycling MDCK cells were trypsinized for 5 minutes in a solution of 0.04% (wt/vol) trypsin and 3mM EDTA to release from culture plastic and collected in 15mL conical tubes
• Cells were centrifuged at 1000 x g for 5 min and re-suspended in low calcium (5µM), low-glucose (200mg/L) DMEM media supplemented with 10% dialyzed FBS at a concentration of 1000cells/µl
• 80-100µl of cell suspension was placed in each strain array well (~80 square mm)
• Cells were allowed to settle for 1 hour in an incubator at 37 degrees C with a 5% CO2 in air humidified atmosphere
• Settled cells were visualized on a cell culture microscope and should appear round due to the lack of adhesion, but touching and uniformly spaced across the bottom of the device well
• To initiate contact formation and adhesion, the low calcium low glucose DMEM was removed and replaced with normal calcium (1.8mM), low glucose DMEM media supplemented with 10% dialyzed FBS
• Cells were returned to the incubator (37 degrees C, 5% CO2 in air, humidified atmosphere) and monolayers were allowed to solidify and mature for 36-48 hours prior to the application of mechanical strain

Preparation of Rat Tail Collagen:

Noda, H. 1960. Physico-chemical studies on the soluble collagen of rat- tail tendon. Biochim. Biophys. Acta. 17:92-98.

Calcium Switch for controlled contact initiation:

Nelson, WJ and Veshnock, PJ. 1986. Dynamics of Membran-Skeleton (Fodrin) Organization during Development of Polarity in Madin-Darby Canine Kidney Epithelial Cells. The Journal of Cell Biology, 103, 1751-1765.