Polyacrylamide gel for Traction force microscopy
Aim
Fluorescent-beads-embedded collagen-coated gel on glass-bottom dish for TFM.
Materials
1ml 3.0% (w/v) acrylamide / 0.25% bis solution (~2 kPa, suitable for MDCK cells)
8 ul APS 10% solution
0.8 ul TEMED
5 ul Fluorescent bead 2% solution (e.g., Invitrogen, FluoSpheres, 0.5 um)
* Bead diameter and concentration are modifieable.
4 mM Sulfo-SANPAH solution (3.94 mg dissolved in 2 ml 0.1M HEPES buffer)
Type-I collagen (0.1 mg/ml)
Methods
Add APS, TEMED, and beads into the acrylamide/bis solution
Put 13 ul of the mixture onto each 35 mm glass-bottom dish with 20-30 mm micro-well.
Put a round cover glass (15mm diameter) onto each droplet.
Invert dishes and incubate them for 60 min at room temperature.
Gently remove the cover glasses
Put 200 ul of Sulfo-SANPAH solution to each dish.
UV radiation (300-370 nm wavelength) for 10 min.
Wash the gels by 0.1 M HEPES buffer
Wash the gels by PBS
Put 1 ml of collagen solution to each dish.
Store them at 4C for >12 hours.
Wash the gels twice by PBS
Put 3 ml culture medium into each dish and incubate them overnight at 37C.
Cell seeding and imaging.
Epithelial spreading assay (optional)
Gently put 2 ul of 8 * 10^6 cells/ml suspension on each gel immersed in medium
Wait for 30 min
Incubate the cells for 48 hours
Imaging
Reference
Ning Wang et al., Am J Physiol Cell Physiol, 2002.
Xavier Trepat et al., Nature Physics, 2009.
DT Tambe et al., Nature Materials, 2011.
Remarks
For convenient measurement of gel stiffness, see (Delanoe-Ayari et al., Cell Motility and Cytoskeleton, 2008).
Date :2019/06/06
Modified Date :
Author : y-kondo