Protocol for PAA gels for traction microscopy

This is a brief description of the Pelham-Wang-Protocol with some variations, mainly for spinning the flurescent beads to the top of the gel to improve resolution. A detailed video protocol can be found under "Video und Erklärungen zu Traction Microscopy (in German only)"

The shear modulus G' of the PAA gels depends on the acrylamide concentration C_ac (in percent, acrylamide:bisacrylamide ratio 29:1 as purchased) according to

G' = 42.82*exp(0.7599*C_ac)

Here are some examples:

  shear Youngs
acrylamide   modulus modulus
concentration   G' E
(%)   (kPa) (kPa)
3.7   0.70 2.11
4.17   1.00 3.01
4.6   1.39 4.16
5.5   2.74 8.22
5.8   3.44 10.31
6.1   4.31 12.93
6.3   5.01 15.04

 

Published data from Yeung et al. (Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil Cytoskeleton 60:24-34, 2005) estimate a somewhat lower value that can be calculated according to

G' = 54 * C_ac^3 * C_bis

whereby both the acrylamide concentration C_ac and the bisacrylamide concentration C_bis are given in %.

 

Preparation of traction gels (E = 12kPa, for other stiffnesses adjust the ratio of water and acrylamide/bisacrylamide according to the table or formulas given above):

  • mix on ice:  417 µl Millipore water with 2.5 µl fuorescence beads (0.2-1 µm);
  • vortex and sonicate for 10 seconds and cool down again. Then add

- 77,6 µl Acrylamide/Bisacrylamide (29:1)
- 2.5 µl APS (10%)
- 1 µl Temed

  • vortex solution and add 28µl to each blue Gene Frame
  • place 170 µm thick activated glass coverslip on frame. Coverslip activation protocol is given below.
  • centrifuge for 30 min, 2500 rpm, 4 C°
  • let polymerize for at least 1h at RT
  • remove plastic film and glue Gene Frame to the bottom of a petri dish with a hole
  • add 2ml PBS and shake for 20 min
  • replace with fresh PBS and place the gel for 1h under UV light to sterilize

    Surface activation with Sulfo-SANPHA and coating with adhesive ligand
  • add 100µl Sulfo-SANPHA solution (0.5mg/1ml PBS) on top of the gel
  • activate for 5 min under UV light (e.g. from the sterile cell culture bench)
  • wash 2x with PBS for 10min on the dish rocker
  • add 500µl fibronectin (10µg/ml) or collagen (50µg/ml) and coat the gel in the fridge overnight
  • wash with PBS and bring the gels to room temperature before seeding cells

 

Activation of glass coverslip (for bonding the PAA gels to the glass)

  • dip cover slips into 0.1N NaOH and let dry for 5 min
  • dip cover slips into 4.0% (3-aminopropyl)triethoxysilane in H2O (prepare solution freshly) and let dry for 5 min
  • dip in Millipore water
  • wash in 200 ml of Millipore water for 10 min on the dish rocker
  • place cover slips in 2.5% glutaraldehyde (in PBS) for 30min
  • wash with Millipore water 2x 10 min on the dish rocker
  • the dried cover slips can be stored in a box at RT

 

Fig: side view of the PAA gels attached to a cell culture dish