Technical Reference #1885

1885.

Filamin A Is Essential for Active Cell Stiffening but not Passive Stiffening under External Force K. E. Kasza; F. Nakamura; P. Kollmannsberger; N. Bonakdar; B. Fabry; T. P. Stossel; N. Wang; and D. A. Weitz, Harvard University, Biophysical Journal, 96(1885), (2009)
Link To Paper

Materials & Methods:
Polyacrylamide gel substrates are prepared according to the procedure described by Pelham and Wang (27) on 35 mm glass-bottomed culture dishes (MatTek Ashland MA). Briefly the glass is aminosilanized to allow for polyacrylamide attachment. Gel stiffness is varied over 2 orders of magnitude by controlling the concentration of the cross-link bis-acrylamide (Bio-Rad Hercules CA) from 0.04% to 0.2% at acrylamide (Bio-Rad) concentrations of 3% 5% and 7.5%. For traction measurements a small volume of 200 nm red latex particles (Invitrogen) is added to the solution to act as a marker of gel deformation. The solution is polymerized on the aminosilanized coverslip by the addition of ammonium persulfate and nnn0n0-tetramethylethylenediamine (TEMED). The polymerizing gel is covered with a second unmodified coverslip and inverted to create a flat gel surface toward which the tracer particles settle. After polymerization is complete the top coverslip is removed and collagen I (Vitrogen; Cohesion Tech Palo Alto CA) at 0.1 mg/mL in solution is chemically cross-linked to the gel surface using sulfo-SANPAH (Pierce Biotechnology Rockford IL). Collagen attachment and uniformity is confirmed using fluorescent collagenfluorescein (Elastin Products Owensville MO). Comparison with known amounts of fluorescent collagen dried on aminosilanized coverslips allows calibration of the collagen surface density to be ~250–650 ng/cm2 for the gels used in this study (28). Gel thickness is 70–100 mm as determined by microscopy. The elastic shear modulus G0 of macroscopic samples of the polyacrylamide gels is characterized in a rheometer (AR-G2; TA Instruments New Castle DE). G0 is related to the Young’s modulus E reported in this work by the Poisson ratio which is taken to be 0.48. Recent work shows that polyacrylamide bulk moduli measured in this way are consistent with atomic force microscopy indentation measurements of polyacrylamide gel substrates (28).

Microscopic Technique
Confocal Microscopy

Cell Type(s)
M2 human melanoma cells;