Hello, has any of you done some research on laminin being as a chemo-attractive protein in cultivating cancer stem cells? Could you share your experience about the work you've done?:)
We are struggling to find the optimum concentration and incubation time of laminin-based-coating and I could really use some advice from more experienced people at this field. We are cultivating the cells in already-coated flasks, but,additionally, we add a coating of poly-L-lysine (1:10) and laminin (1:100), but the cells seem to adhere unevenly and differently.
Thank you for replies!
A lab I was formerly in did adhesion assays with increasing concentrations of cell attachment substrates in order to determine what concentration gave maximal adhesion. This was with rat chondroprogenitors and differentiated chondrocytes, but the same approach could work for testing laminin. The paper is available on ResearchGate: [Fernandez C, et al. (2010) Recognition of the alternatively spliced segments of fibronectin by the RCJ3.1C5.18 chondrocytic rat cell line. Osteoarthritis Cartilage 18: 228-39]. Poly-L-Lysine isn't included in the experimental results, but we found the maximal adhesion concentration (for PLL) to be 1 ug/ml. This was a dilution of 1:100 in DMSO from an original stock concentration of 1 mg/ml (if memory serves).
It has been shown that cancer cells show the deposition of laminin gamma-2 chain when they undergo EMT process, in which the number of CSCs dramatically increases. Indeed, 3D culture system using laminin leads to the increased expression of "functional cancer stem cell markers" such as EpCAM and CD44 variant.
We work with a laminin receptor and find the absolute best source of laminin-1 is Trevigen (they are also the cheapest!). To coat any plastic surface, you can dilute your laminin in PBS at 10 micrograms per ml and let it sit on the plastic for 1 hr. You can also reuse this solution for other plates. Then add your cells in their medium to culture. As Go says above, many cells will deposit their own laminin, often laminin-5 (also known as laminin 332, which is defined by the unique gamma-2 chain). I'm a little unsure why you are adding the poly-L lysine. Do you just need cells to adhere? Or is this to reduce the cost?
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