Sep 15, 2022

Public workspaceProtocol for mixed cortical striatal cell culture 

DOI
dx.doi.org/10.17504/protocols.io.j8nlkw5y1l5r/v1
  • 1Northwestern university
divya.darwinarulseeli
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DOI: dx.doi.org/10.17504/protocols.io.j8nlkw5y1l5r/v1
Protocol Citation: Chuyu Chen, Ciarra Smith, Loukia Parisiadou 2022. Protocol for mixed cortical striatal cell culture . protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkw5y1l5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 15, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70097
Keywords: ASAPCRN
Abstract
This protocol describes a optimised protocol for culturing cortical and striatal neurons from postnatal pup brains
Materials
Solutions Needed
  • Poly-D-lysine
oStock: 200mg/ml
oDilute to 50µg/ml for coating
  • AraC 1000x
  • Basal Medium Eagle (dissection medium, nothing added, on ice)
  • Basal Medium Eagle + supplement (water bath, used for initial incubation)
  • Basal Medium – supplement + RFC serum
  • Papain Enzyme
Add 500µl of poly-D-lysine solution to each well that will be used for plating
Allow to sit inside the hood for at least 30mins (this plate will stay under the hood until dissection is finished)
Set BME + supplement and BME+AraC serum in water bath
Add 1-2ml of BMS alone into 2 wells of a 24-well plate (not coated)
Add 4-5ml of BMS alone into 16mm culture dish
Place dish on ice and leave BME inside the hood
Dissect P0/P1 brains on ice in to dissection medium
Aspirate medium gently, add 1ml of enzyme and incubate for 20 minutes at 37 degrees
Note: It is better to cut tissue sample using scalpel to allow more area for optimal enzymatic digestion
Wash 3X with 1ml of room temp BME medium without supplement to wash out the enzyme
Add 2ml of medium to well
Gently pipette 15 times and transfer to 15ml tube
Wait ~1min for large chunks to settle
Carefully remove supernatant and transfer to fresh 15ml tube
Centrifuge: 300g, 4°C, 4min
Remove media and resuspend in 1ml of media + serum
Remove poly-d-lysine from 24-well dishes
Add 500µl of medium + serum to each well
Count cells: 5x10^5 cells/well
Plate 1:2 striatum:cortex
Incubate for 1hr then change media
Add GDNF to BME-(without serum; 1µl of GDNF in 2.5ml)
Incubate cells in a CO2 incubator