Technical Reference #1919

1919.

Extracellular Matrix Stiffness and Architecture Govern Intracellular Rheology in Cancer Erin L. Baker; Roger T. Bonnecaze; and Muhammad H. Zaman, University of Texas at Austin, Biophysical Journal, 97(1919), (2009)
Link To Paper

Materials & Methods:
First MatTek product - For 2D matrices 2.9 mg/mL Type I collagen (BD Biosciences San Jose CA) was diluted to three concentrations of 25% (0.73 mg/mL) 37.5% (1.09 mg/mL) and 50% (1.45 mg/mL) v/v with F-12K media; 20 mL of each solution was then evenly spread across the glass-bottom surface (10 mm) of a 35-mm dish (MatTek Ashland MA) and exposed to ammonia vapors to promote rapid gelling of the collagen coating across the dish surface. For 3D matrices 2.9 mg/mL Type I collagen was diluted to concentrations of 25 37.5 and 50% v/v with cells suspended in complete F-12K media. Each solution (100 mL) was then deposited onto the glass-bottom surface of a 35-mm dish and allowed to gel for 6 h at 37 C 5% CO2. Second MatTek product - Two-dimensional PA gels of varying stiffness were prepared as described previously (20). Briefly a mixture of acrylamide and bis-acrylamide solutions were polymerized with TEMED (Thermo Scientific Pierce Milford MA) and 10% ammonium persulfate. Acrylamide concentration was held constant at 3% with bis-acrylamide concentrations fixed at either 0.04 0.06 or 0.2%. A total PA solution volume of 20 mL was allowed to polymerize across the glass-bottom surface (30 mm) of a 50-mm dish (MatTek Ashland MA). Before depositing the unpolymerized PA solution on the glass-bottom surface the surface was sequentially pretreated with 0.1 N NaOH 3-APTMS (Sigma St. Louis MO) and 0.5% glutaraldehyde (20). Upon depositing the unpolymerized PA solution on the surface the solution was flattened using a 22-mm circular glass coverslip (Fisher Scientific Pittsburgh PA). Once the polymerization was complete the coverslip was carefully removed and the surface of the 2D PA gel was cros-slinked with 25% v/v (0.73 mg/mL) Type I collagen. This method yields PA gels of stiffness 50 200 and 400 Pa (20) (with respect to the bis-acrylamide concentrations noted above) and constant collagen ligand density across the surface (21).

Microscopic Technique
Confocal Microscopy

Cell Type(s)
PC-3 cell line