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:
- Under sterile conditions, dilute the 1 mg/mL PLO stock to a final concentration of 50 µg/mL (1:20 dilution) in sterile PBS.
- Add the PLO solution to your culture vessel.
- Incubate the plates at 37°C overnight.
- Wash 3 times with sterile PBS before coating with laminin.
Laminin Coating:
- Thaw working aliquot of laminin slowly on ice. Avoid formation of a gel, as laminin cannot be reactivated if allowed to gel.
- 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).
- Add the laminin solution to your PLO coated culture vessel.
- Incubate the plates at 37°C for 1 hours.
- 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