Dear Olga,

The only times I have had laminin precipitate out on my plates is when there is calcium or magnesium present in the PBS, if the plates have dried out, or the laminin was poorly handled when aliquoted for storage.

I know it seems that everyone has their own coating procedure.  I usually coat my plates first with Poly-D-Lysine in 0.1M borate buffer pH 8.3 overnight, rinse extremely well (4x with sterile water, then 2x with Dulbecco's PBS that lacks Calcium and Magnesium).  I then coat with my laminin dissolved in the calcium- and magnesium-free Dulbecco's PBS overnight, and rinse 5x with the same PBS, then one time with the media before I plate my cells.  For 35mm plates I usually use a coating volume of 1.5ml, and rinse with 2 ml.  I have also coated TC plastic with laminin only, but for neurons I always use the PDL first, so that I can bind a lot of laminin to the plate (more charge = more capture of the laminin).  However, I study axonal outgrowth from adult neurons; your cells may not need as much laminin.

Another precipitation problem can arise when you aliquot the laminin for storage.  We always thaw the initial stock vial at 4 C (see product sheet below), and make sure it is liquid before aliquoting into single use size.  We make sure the tubes for the aliquots are on ice in the hood, and keep the laminin on ice also.  We use a Pipetman that has a multiple aliquot mode, and make sure to cap each tube as it is filled.  If you let the small aliquots sit in laminar flow without caps, they can dry out and become "pre-polymerized" if you will.

Even if I am really careful, I always check my plates for precipitate before i even start dissecting the tissue to obtain the neurons.  There have been a few unpleasant surprises over the years.  For me, the tissue is much more valuable than the laminin.

Good Luck!

Jill

https://tools.thermofisher.com/content/sfs/manuals/naturalmouselaminin_man.pdf

Well... evidently, the impurities: such as entactin are critical for some cell culture adhesion. As a result, ultra-pure versions of it are not suitable for the ECM for those cultures.