What concentration of Laminin is ideal for neuronal culture? And should I dissolve it in water or PBS?
Compared to collagen/gelatin coatings it is crucial that laminin coatings should not dry out for a good cell attachment...
HI Pranay, I 've seen different concentrations in several protocols from different labs, ranging usually from 5 to 50 ug/ml. Most of the time Laminin is added on top of a PLL pre-coating which might range from 10ug/ml to 1mg/ml. Concentration depends on the type of neuron you'll study as well as the type of experiment you want to perform.
I generally use 1mg/ml PLL coating and 5ug/ml of Laminin on top. Other concentration found in many papers uses 50ug/ml of PLL pre-coating and 25 ug/ml of Laminin (used quite often on estudies that investigate formation/stability of adhessions).
I like dissolving the Laminin in PBS.
I'm sure you'll get plenty of suggestions.
Hope it helps,
Dear Pranay,
I suggest that you check the literature for the type of neuron you wish to culture, as well as determine whether the investigators simply applied the laminin to plastic or glass, or whether they first coated the plastic or glass with Poly-D-Lysine (or Poly Ornithine). Since the lab in which I work is interested in the role of extracellular matrix proteins in adult CNS axonal outgrowth and regeneration, and we culture on glass in 35 mm dishes, I have always coated first with 0.5 mg/ml of Poly-D-lysine HBr (>300,000 MW, Sigma) in 0.1 M Borate Buffer, pH 8.3, followed by 5 ug of mouse EHS laminin* in 1.5 mls of Ca++/Mg++ FREE PBS (per 35 mm dish). You must rinse the plates very well between the PDL coating and the laminin coating--I rinse 6 times.
I have always diluted laminin in Dulbecco-Vogt PBS that doesn't have calcium or magnesium added to it, since I have had the laminin precipitate out in the presence of these ions. You can't necessarily see it in solution, but you can see it under phase microscopy on the surface of the plates or coverslips.
My recipe for 1 liter is:
NaCl 8.2 g
KCl 0.2 g
Na2HPO4 (anh) 1.15 g
KH2PO4 0.2 g
pH 7.3-7.4; sterilize by filtration; stable at room temperature.
For convenience, I coat with each solution overnight. However, be very careful to prevent the laminin from drying out. Drying can change the conformation of the laminin molecule so that it doesn't support good axonal outgrowth. It is also inactivated by UV light, so don't expose your plates or wells to UV lamps.
Good Luck!
Jill
Neurons grow on 0.1-1 mg/ml poly d lysine substrate. You can add laminin depending neuron type. Mouse central neurons require only poly lysine, whereas rat neurons may benefit from laminin addition. DRG strictly require laminin.
If you are coatingwith polylisine alone be careful to wash with water and let dry before seeding cell, if laminin is present wash with pbs and aspirate before seeding cells, do not let dry.
Thanks Sebastian I followed your suggestion and found the concentration that I need for my purpose.
Thanks a lot Jill for your detailed suggestion, since now I know what concentration to use I will follow the recipe that you are using.
Compared to collagen/gelatin coatings it is crucial that laminin coatings should not dry out for a good cell attachment...
I use 20ug/mL laminin (Millipore) solution and coat overnight at 4oC. Once removing the solution from the plate I retain that and reuse only once more. As mentioned by Johannes laminin must not dry, and also it is best if coated plates are kept in the fridge.
We usually use 10-100 uL/mL for neuronal coatings. We sterilize at room temperature for 30 min. I would also recommend to incubate in the cold. For pilots, we re-use the laminin up to 3 times, but not for studies (loss of bioactivity was observed). All the best!
Johannes,
What do you mean you 'sterilize at room temperature for 30 minutes'?
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