Poly-ornithine/laminin substrate for neural cell culture
Regine Tipon, Gist Croft
Abstract
This protocol is used to create adhesive and bioactive substrate for neural cell types, low to high density nuerons, astrocytes, or organoids. It is based on standard methods but includes several optimizations, use case recommendations, and alternatives, and advice.
Attachments
Steps
Preparing Reagents
Dissolve unopened vial of Poly-Ornithine in Borate Buffer at 1mg/ml (P-Orn)
Filter sterilize and store at 4 degrees for 3 months
Pretreatment for coverslips:
Sterilize forceps in 100% EtOH
Dip 15mm coverslip generously in 100%EtOh, place in 24wp or 4wp wells
Wash wells 3x with TC H2O
Aspirate completely and Dry in hood
Coating
Add P-Ornithine solution to cover well
~0.5ml/24wp well or 8 well slide well
Note: if using coverslip, make sure to tamp down coverslip with sterile tip
Incubate 2-6 hrs to overnight in incubator
Add P-Ornithine solution to cover well
Wash wells 2-3x, 0-2 min each with ≥coating volume of TC grade H2O
borate+polyamino acid solution is toxic, aspirate completely during washes and use greater than coating vollume for washes.
When ready to seed cells, aspirate laminin (can save for reuse) and seed cell solution directly without drying substrate
Add Laminin in L15+ NaBicarbonate solution: final 10ug laminin/ml, same volume and incubate ≥ 12 hours in TC incubator
As long as laminin solution does not dry, substrate is good for several weeks in incubator, add water each week.
Alternatively, desalt wash 2-3x with H2O and dry (see Drying Protocol below).
Best to dry freshly prepared substrate, rather than one that has been incubating for some time
Drying coated surfaces
Aspirate laminin
Wash 3x with TC water
Completely aspirate water, including residual droplets, without scratching surface
Dry dishes open in the hood for 15-20 min.
Parafilm and store at 4 degrees for 6 months
