Rat collagen type 1 solution

Confocal experiments were prepared by mixing imaging media and non-pepsin-treated rat collagen type I solution (Corning, Corning, NY, USA) to a concentration of 2 mg ml⁻¹. A volume of collagen solution (125 μl) was pipetted into a MatTek dish. MatTek dishes are conventional 35 mm tissue culture dishes with a hole in the bottom that is covered with a glass coverslip. This coverslip is glued to the bottom, creating a shallow well (MatTek Corporation, Ashland, MA, USA). Collagen is spread evenly on the bottom of the well, and allowed to polymerize for 45 mins. A 0.25 × 25 mm stainless steel acupuncture needle with a tapered tip (Hwato, Weymouth, MA, USA) was inserted through a 5 mm thick pad of polydimethylsiloxane (PDMS) (Dow Corning Corporation, Midland, MI, USA) to the depth of the well and placed on top of the collagen gel, sealed with food grade lubricant (The McGlaughlin Oil Company, Columbus, OH, USA). The needle was then rotated the required degrees slowly to limit the amount of gel tearing and cut with wire cutters.

Cell migration experiments were conducted by mixing trypsinized cells at a concentration of at 600,000 cells ml⁻¹ in a 2 mg ml⁻¹ non-pepsin-treated rat collagen type I solution (Corning, Corning, NY, USA). The protocol is similar to the confocal experiments, however after polymerization the well is filled with 125 ml of imaging media specific to the cell type. The needle is either not rotated or rotated one full rotation (2π) and then cut by wire cutters. The samples are then placed in a 37 °C incubator. After 24 hr the samples are moved to a heating stage to keep the samples at 37 °C and imaged every 2 min for 8 hr. At least 3 samples over at least 2 different days compiled a complete data set.