Coating plates/wells with laminin. - (Oct/17/2013 )
How does one determine what is the best concentration of laminin when coating 24 well plates? I have seen people use 20ug/ml, 50ug/ml or even 1-2 ug/cm2. Also, what happens to the cell viability if you use too high of a concentration of laminin? Does it have any effect at all?
Btw, lamini starts to polymerize and become gel-like above 4 degrees, what if you freeze it again? Would it depolymerize?
It really depends on what experiment or what you want to use laminin for. Most people use it to improve cell attachment for fragile cells (neuronal cultures) or it's just the standard protocol to culture a certain cell type.
I've used laminin concentrations of up to 100ug/ml and the cells totally love it. Do take note that there will be an absolute maximum of substrate which you can coat the surface area with as most excess will be washed away anyway. What cells are you intending to culture in those plates?
It is best to thaw any ECM substrate slowly on ice and reduce harsh pipetting. It's also good to avoid vortexing all together as it will disrupt the protein structure. I suggest you aliquot your laminin stocks (normally comes in a 1mg/ml stock) into small microfuge tubes (you will need to plan the volumes per aliquot depending on how frequent you will need them and how much you will need each time). For me, I will avoid repeated freeze thawing of my aliquots. I will use only the freshest aliquots for my important experiments.