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.