|Product Name||Format||Stock Conc.||Storage on Arrival||Thawing Instructions||Storage Once Thawed|
|ax0091||Human iPSC-Derived Dopaminergic Neuron Progenitors||1 million cells/vial||N/A||Liquid Nitrogen||Follow protocol||N/A|
|Dopaminergic Neuron Basal Medium||50 mL||1x||-20ºC||Overnight at 4ºC||Store at -20ºC for up to 6 months|
|Supplement A||1 vial 80 μL||1x||-20ºC||Overnight at 4ºC||Store at -20ºC for up to 6 months|
|Supplement B||1 vial 60 μL||1x||-20ºC||Overnight at 4ºC||Store at -20ºC for up to 6 months|
|ax0041+||SureBond||3 x 120 μL||50x||-80ºC||Overnight at 4ºC||Store at 4ºC for up to 2 weeks|
|ReadySet||2 x 10 mL||1x||4ºC||N/A||Store at 4ºC for up to 1 month|
Lot-specific information such as specifications and quality control details are stated in the Certificate of Analysis.
- Recommended culture vessecoating: SureBond+ReadySet
- Recommended celculture medium: Differentiation Medium for 5 days followed by Maintenance Medium
- Recommended seeding density for assay: 40,000-60,000 viable cells/cm2
- Recommended centrifugation speed: 400 x g for 5 minutes
- Recommended days in culture before assay: 14 days (minimum for tyrosine hydroxylase expression) (5 days in Differentiation Medium then 9 days in Maintenance Medium)
Cardiomyocyte Maintenance Medium = Basal medium + Supplement DOES NOT contain antibiotics or antifungal agents. Axol Bioscience does not recommend the use of antimicrobial agents such as penicillin, streptomycin and amphotericin. Antimicrobial agents should not be necessary if proper aseptic technique is adopted.
Dopaminergic Neuron Differentiation Medium
- Upon receipt aliquot and store the Dopaminergic Neuron BasaMedium at or below -20°C protected from light. Make 1 x 20 maliquot and 2 x 15 maliquots. Stored at -20°C, the medium is stable for 6 months from date of manufacture.
- When ready to use, thaw the 20 maliquot of BasaMedium overnight at 4°C in the dark.
- On the day of thawing Human iPSC-Derived Dopaminergic Neuron Progenitors, transfer 20 mof aliquoted BasaMedium to a fresh 50 mtube.
- Pre-thaw Supplement A on ice and add the entire contents (80 μL) to the 20 maliquot of BasaMedium. This will make Differentiation Medium.
Dopaminergic Neuron Maintenance Medium
- When ready to use, thaw a 15 maliquot of BasaMedium overnight at 4°C in the dark.
- On day 5 post-seeding, transfer 15 mof aliquoted BasaMedium to a fresh 50 mtube.
- Thaw Supplement B on ice and add 30 μto the 15 maliquot of BasaMedium. This wilmake Maintenance Medium.
- Refreeze the remaining Supplement B (30 μL) untimore Maintenance Medium is required.
Important! Axol recommends: SureBond+ReadySet coating reagent for Human iPSC-Derived Dopaminergic Neuron Progenitors.
Thawing and Plating Human Dopaminergic Neuron Progenitors
- On the day before thawing Human Dopaminergic Neuron Progenitors cells, prepare the Differentiation Medium.
- Pre-warm almedia and vessels to 37ºC before use.
- Aliquot 5 mof Differentiation Medium into a 15 msterile tube and pre-warm at 37ºC.
- Prepare a second tube with enough Differentiation Medium for celculture vessels (see Table 1) and pre-warm at 37ºC. Store the remaining medium at 4ºC.
- To thaw cells – transfer the viaof cells from liquid nitrogen storage with the viaburied in dry ice. Remove the vial from dry ice and transfer it immediately to a 37ºC water bath.
- Quickly thaw the viaof cells by swirling it in a 37ºC water bath. Do not completely submerge the via(only up to two thirds of the vial). Remove the viabefore the last bit of ice has melted, after ~1-2 minutes.
- Do not shake the viaduring thawing.
- Take the viaof cells to a biologicasafety cabinet, spraying it thoroughly with 70% ethanoand wiping with an autoclaved paper towebefore placing it in the hood.
- Once thawed, use a P1000 pipette to transfer the cells drop-wise into a 15 msterile conicatube containing 5 mL of pre-warmed (37ºC) Differentiation Medium. Gently wash the viawith 1 mof Differentiation Medium. Transfer
- this to the 15 msterile conicatube containing the cells.
- Centrifuge cells at 400 x g for 5 minutes at room temperature Aspirate the supernatant carefully and resuspend the celpellet with 1 mof Differentiation Medium.
- Gently resuspend the cells untithey are in a single celsuspension.
- Remove 10 μof celsuspension and mix it with 10 μof trypan blue solution. Count the cells.
- Calculate the appropriate volume of Differentiation Medium with respect to the culture vesse(see Table 1).
- Resuspend the cells in Differentiation Medium and plate the resuspended cells at a density ranging from 40,000 – 60,000 cells/cm2 on the SureBond+ReadySet coated culture vessel.
- Plate the cells drop-wise and evenly on the culture vessels. Incubate the cells at 37ºC, 5% CO2.
- After three days, replace the medium with fresh, pre-warmed, 37ºC, Differentiation Medium. Medium should be
- changed gently in a drop-wise fashion while pointing the pipette tip toward the walof the culture vessel. Refresh the medium every 2 days.
Important! Do not mix the cells vigorously. Avoid generating bubbles
|Vessel Type||Medium Volume|
|96-well plate||100 μL/well|
|24-well plate||500 μL/well|
|35 mm dish||2 mL|
|60 mm dish||5 mL|
Maintenance and Maturation of Human Dopaminergic Neuron Progenitors
- On day 5 post-seeding, prepare the Maintenance Medium and pre-warm (37ºC) an aliquot. Store the remaining medium at 4ºC.
- Gently replace the Differentiation Medium with Maintenance Medium.
- Continue maturation of dopaminergic neurons in Maintenance Medium. Change the medium every 2 days.
- Dopaminergic neurons can be stained and analysed 14 days after seeding.
- After 14 days in culture, 30% of the dopaminergic neurons should be tyrosine hydroxylase positive
Top Tip: Dopaminergic neurons should be matured for up to 5 weeks after seeding in order to identify dopaminergic neuron receptors and to obtain electrically active dopaminergic neurons. Longer culture will also increase population of astrocytes (GFAP positive).