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PLO/Laminin Coating Protocol

Laminin is an extracellular matric (ECM) protein and a key component of the basal lamina of epithelial tissues in the body. After an initial coating poly-ornithine or dPGA, it is often necessary to add a second layer of laminin to your in vitro culture system to support the adhesion, proliferation and differentiation of many cell types including endothelial, epithelial, neural, liver and muscle cells. 

Materials:

  • Poly-L-Ornithine hydrobromide (PLO) - Sigma Aldrich #P3655
  • Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane- Sigma #L2020
  • PBS

Storage/Stability

  • Store desiccated PLO powder or stock solution at -20°C.
  • Store undiluted Laminin at -20°C until use.

Stock Preparation

  • Under sterile conditions, prepare stock solutions of 1 mg/mL PLO in sterile ddH2O. Solution should be clear. Filter sterile with a 0.22µm Teflon filter. Store aliquots at -20°C.
  • Thaw the laminin solution slowly on ice to avoid formation of a gel. Under sterile conditions, prepare working aliquots and store at -20°C. 

    PLO Coating:

    1. Under sterile conditions, dilute the 1 mg/mL PLO stock to a final concentration of 50 µg/mL (1:20 dilution) in sterile PBS.
    2. Add the PLO solution to your culture vessel.
    3. Incubate the plates at 37°C overnight. 
    4. Wash 3 times with sterile PBS before coating with laminin. 

    Laminin Coating:

    1. Thaw working aliquot of laminin slowly on ice. Avoid formation of a gel, as laminin cannot be reactivated if allowed to gel. 
    2. Dilute the laminin stock to desired concentration in sterile PBS. The ideal concentration is application-dependent (but a typical concentration to start from is 1 µg/mL). 
    3. Add the laminin solution to your PLO coated culture vessel. 
    4. Incubate the plates at 37°C for 1 hours. 
    5. Wash 3 times with sterile PBS before plating cells. Keep the coated surface in PBS, do not allow the surface to dry for more than a few minutes. 

    References

    Clément, J.-P., Al-Alwan, L., Glasgow, S. D., Stolow, A., Ding, Y., Quevedo Melo, T., Khayachi, A., Liu, Y., Hellmund, M., Haag, R., Milnerwood, A. J., Grütter, P., & Kennedy, T. E. (2022). Dendritic Polyglycerol Amine: An Enhanced Substrate to Support Long-Term Neural Cell Culture. ASN Neuro, 14, 17590914211073276. https://doi.org/10.1177/17590914211073276