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
dh4fbiqa7.pdf
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.
