PEI/Laminin Coating
Zoe
Abstract
This protocol is used to prepare plates for iPSC-derived neuron maturation. It is a part of the NGN2 cortical neuron differentiation protocol: coat plates in preparation for Day 7 dissociation and replating.
Attachments
Steps
PEI Coating
At least one day before plating, make 2x borate buffer with 4mL 20x borate buffer (Thermo, cat. no. 28341) in 36mL water in a 50mL Falcon tube.
Dilute 800µL 5% PEI (poly-ethylenimine; stored at 4°C) in 40mL 2x borate buffer to make 0.1% PEI.
Filter-sterilize with SteriFlip: attach filter and tube to 50mL tube with PEI solution; flip and attach to vacuum; when medium drains through, remove filter and original tube.
Add 150µL to each of the inner 60 wells of a 96 square-well plate (Brooks, cat. no. MGB096-1-2-LG-L); 500µL for wells of 24-well plate (Corning, #3527).
On 96-well plates, leave a border of empty wells to avoid edge effects; fill these with PBS to maintain humidity.
Label plate(s), wrap sides with parafilm and cover in plastic wrap; store at 4°C .
Laminin Coating
On the day of passaging, make laminin 5µg/ml in 4°C PBS (with calcium and magnesium ions), ex. 1mL PBS with 5µL laminin (Sigma, cat. no. L2020, 1mg/mL).
Wash PEI-coated plates twice with distilled water (300µL for 96-well plates).
Wash once with PBS.
Add 150µL laminin to wells of 96-well plate, and 0.5mL to wells of 12-well.
Incubate plates at least 2 hours at 37°C before re-plating neurons.
