Fully-defined system to synchronously
Preparation of Plating-XF Medium
- 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.
- When ready to use, thaw plating media overnight at 4°C in the dark.
- 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)
- Check the total number of viable cells on the cryovial or on the Certificate of Analysis shipped with the cells.
- 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).
- 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.
- 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
- 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
- Remove the cells from dry ice or liquid nitrogen storage. Immediately transfer the cells to a 37°C water bath.
- 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.
- When thawed, immediately transfer the cells into a 15 mL sterile conical tube, and carefully add 10 mL of Axol Plating-XF Medium.
- Centrifuge the cells at 200 g for 5 mins, and discard the supernatant.
- Resuspend the cell pellet in the required amount of Axol Plating-XF Medium. .
- We recommend the use of 200 μl Axol Plating-XF Medium per cm 2 .
- Quickly remove the diluted Axol SureBondXF coating solution from the pre-coated culture vessel before plating resuspended cells.
- Plate the resuspended cells with Axol Plating-XF Medium according to density guidelines (cell line dependent) on your Axol SureBondXF coated culture vessel.
- Incubate the plated cells at 37°C, 5% CO 2 .
- 24 hours after plating, replace the spend medium with fresh pre-warmed Axol Neural Differentation-XF Medium.
- After 72 hours, replace half the volume of spent medium with fresh, Axol Neural Maintenance-XF Medium.
- 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!