Fabrication of gel beds

Fabrication of gel beds.
The fabrication protocol for the Geltrex gel bed was based on a ‘sandwich’ set-up previously developed for generating polyacrylamide gel substrates1,2,3.

The detail fabrication scheme is as follows：

1. 22×22 mm² glass coverslips were sterilized.
2. This step aims to prepare a coverslip that the gel bed could attach.
   Treating a 22×22 mm² glass coverslip with air plasma (Harrick Plasma) for 2 min. Then coating the coverslip with 0.1 mg/ml poly-(L-lysine) (PLL) solution (Sigma- Aldrich) for 30 min. Removing the PLL solution completely and coating the coverslip with 1% glutaraldehyde solution (Electron Microscopy Sciences) for another 30 min.
3. This step aims to prepare a coverslip that the gel bed was easy to separate from the coverslip.
   To prepare a coverslip that could ‘sandwich’ and release the gel bed, a pre-cleaned 22×22 mm² glass coverslip was treated with air plasma for 2 min and then was coated with 0.1 mg ml−1 poly-(L-lysine)-graft-poly-(ethylene glycol) (PLL-g-PEG; SuSoS) solution for 1 h.
   Steps 2 and 3 should be done at the same time.
4. To obtain gel beds with nominal thicknesses of 100 µm, 50 µl undiluted Geltrex was sandwiched between the above two pre-treated coverslips. Then transferring the sandwiched coverslips into cell incubator and incubated at 37 °C for 30 min (The time may need to be extended according to the researcher's operation) for gelation. The gel bed, which was attached to the glass coverslip in step 2, was then removed from the glass coverslip in step 3 and submerged in DMEM/F12 medium (Thermo Fisher Scientific) and incubated at 37 °C overnight before platingcells.

Company name and catalog number of some reagents:

GeltrexT (Thermo Fisher Scientific)                                                          A1413202

Advance DMEM/F12 (Gibco)                                                                   12634010

poly-(L-lysine) (sigma)                                                                             P4707-50ML

Glutaraldehyde stock, 25% (wt/vol) (Electron Microscopy Sciences)     16200

poly-(L-lysine)-graft-poly-(ethyleneglycol) (SuSoS)                                PLL(20)-g[3.5]- PEG(2)

References

1. Buxboim, A., Rajagopal, K., Brown, A. E. X. & Discher, D. E. How deeply cells feel: methods for thin gels. J. Phys. Condens. Matter. 22, 194116 (2010)
2. Tse, J. & Engler, A. Current Protocols in Cell Biology Preparation of hydrogel substrates with tunable mechanical properties. Ch. 10 (Wiley, 2010).
3. Fischer, R., Myers, K., Gardel, M. & Waterman, C. Stiffness-controlled three-dimensional extracellular matrices for high-resolution imaging of cell behavior. Nat. Protoc. 7, 2056–2066 (2012).