Investigating Axonal Biology Using Cortical Neurons with the Neurite Outgrowth Microfluidic Device
Cells and Reagents
- Cortical Neuron Differentiation Kit -includes Neural Enhance, Sure Boost and Neural Stem Cells (ax0025f or ax0026f)
- Neural Maintenance-XF Medium (ax0030-500)
- Axol Sure Bond+ - Includes Axol Sure Bond and Axol ReadySet (ax0041+)
- Neurite Outgrowth Microfluidic Device (ax5000)
- 24 mm x 40 mm No.1 cover glass (Corning, Thermo or alike
- Sterile dH2O
- 70% Ethanol
- D-PBS (without calcium or magnesium)
- 60 mm tissue culture dish
- Water bath sonicator
- 37°C 5% CO2 tissue culture incubator
Preparing Cover Glass for Device Attachment
1. Use a water bath sonicator to clean the cover glass to be used for growing neurons. This can be done by placing the cover glass in a rack filling it with dH2O and sonicating for 30 minutes.
2. While the cover glasses are being cleaned in the sonicator, place your Axol ReadySet solution in the tissue culture hood.
3. Transfer clean cover glass to the tissue culture hood to maintain sterility and then rinse the cover glass with 70% Ethanol.
4. Rinse the cover glass 3x with sterile dH20.
5. Submerge cover glass in Axol ReadySet solution. Allow the cover glass to be coated for at least 4hr or overnight at 37ºC.
6. Thaw the Axol Sure Bond coating solution overnight at 4°C.
7. Wash the cover glass 4 times using an equal volume of dH2O (e.g. if 250 µL Axol ReadySet, use 250 µL ddH2O per wash).
8. Clean and sterilize the device with 70% Ethanol using a spray bottle. Allow the device to air dry in the tissue culture hood for 1hr with feature side up.
9. Dilute the Axol Sure Bond stock solution (50X) in D-PBS (without calcium or magnesium) to make 1X working solution e.g. 120 μL in 6 mL.
10. Coat the cover glass with the Axol Sure Bond 1X working solution. We recommend coating at a concentration of 200 μL per cm2, however, please optimize for your experiments.
11. Incubate for 1 hour at 37ºC