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Differentiation of Human Neural Progenitor Cells Using Axol’s Cortical Neuron Differentiation Kit Protocol

Product Information

Fully-defined system to synchronously

Preparation of Plating-XF Medium

  1. Upon receipt, store Axol Plating-XF Medium at or below -20°C protected from light. Stored at -20°C, media is stable for 6 months from date of manufacture.
  2. When ready to use, thaw plating media overnight at 4°C in the dark.
  3. Once thawed, Axol Plating-XF Medium should be used immediately and should not be used for subsequent experiments.

Preparing Matrix for Adherent Cell Culture (ax0041XF)

  1. Check the total number of viable cells on the cryovial or on the Certificate of Analysis shipped with the cells.
  2. Calculate the total surface area that requires coating. This is the total number of viable cells (e.g. 2 million) / your desired plating density (see page 4 for guidelines).
  3. Dilute the Axol SureBondXF stock solution (200X) in D-PBS (without calcium or magnesium) to make 1X working solution e.g. 30 μL in 6 mL.
  4. Coat the surface of your culture vessel with the Axol SureBondXF 1X working solution. We recommend coating at 200 μL 1X solution per cm 2
  5. Incubate for 4 hours at 37ºC.

Warning: Do not wash the vessel after coating with Axol SureBondXF. Do not allow Axol SureBondXF -coated culture vessels to dry.

Thawing Axol NSCs for Synchronous Differentiation

  1. Remove the cells from dry ice or liquid nitrogen storage. Immediately transfer the cells to a 37°C water bath.
  2. Quickly thaw the vial of cells by swirling it in the 37°C water bath. Do not completely submerge the vial. Remove the vial before the last bit of ice has melted.
  3. When thawed, immediately transfer the cells into a 15 mL sterile conical tube, and carefully add 10 mL of Axol Plating-XF Medium.
  4. Centrifuge the cells at 200 g for 5 mins, and discard the supernatant.
  5. Resuspend the cell pellet in the required amount of Axol Plating-XF Medium. .
  6. We recommend the use of 200 μl Axol Plating-XF Medium per cm 2 .
  7. Quickly remove the diluted Axol SureBondXF coating solution from the pre-coated culture vessel before plating resuspended cells.
  8. Plate the resuspended cells with Axol Plating-XF Medium according to density guidelines (cell line dependent) on your Axol SureBondXF coated culture vessel.
  9. Incubate the plated cells at 37°C, 5% CO 2 .
  10. 24 hours after plating, replace the spend medium with fresh pre-warmed Axol Neural Differentation-XF Medium.
  11. After 72 hours, replace half the volume of spent medium with fresh, Axol Neural  Maintenance-XF Medium.
  12. After 24 hours, replace half the volume of spent medium with fresh, pre-warmed Axol Neural Maintenance-XF Medium. Repeat this process of re-feeding cultures with half-volumes of fresh, pre-warmed Axol Neural Maintenance-XF Medium every four days.

Top Tip: It is critical to promote consistent cell density, monolayer and health throughout the culture to avoid edge effects and variations in cellular maturity. After seeding, avoid disturbing the culture vessel for a minimum of 30 minutes to allow the cells to adjust to their environment.

Top Tip: Cultures can be maintained under these conditions for over 50 days in culture!