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Description
Product Specification
| Starting material | Dermal fibroblast |
| Donor gender | Female |
| Donor age at sampling | 64 yrs |
| Karyotype | Normal |
| Reprogramming method | Episomal vector |
| Induction method | Monolayer & chemically defined medium |
| Genetic modification | Homozygous for the LRRK2 G2019S mutation (GGC>AGC) |
| Genetic modification | Contains a puromycin resistance cassette (intronic) |
| Size | ≥1.5 million cells |
| Kit components | 1 vial of axolGEM Neural Stem Cells (≥1.5 million cells) and 1 bottle of Plating-XF Medium (30 mL) |
| Growth properties | Adherent |
| Shipping conditions | Dry ice |
| Storage conditions | Liquid nitrogen |
Frequently Asked Questions
I want to carry out imaging/confocal microscopy, how should I plate the cells?
Plate the cells on Readyset + Surebond (ax0052)
I want to passage and expand the iPSC-derived neural stem cells, what coating shall I use?
Always plate onto Surebond (ax0041) if you intend to detach the cells for continued expansion
I am not sure what coating I should use for iPSC-derived neural stem cells?
Surebond-XF - Xeno-free coating reagent needed for endpoint assays on plastic, this is the easiest method of coating, with no wash steps. Surebond + Readyset - Coating reagent needed for endpoint assays on glass or 96-well plates Surebond- Coating reagent needed when passaging of NSCs is required
For cortical neurogenesis, when do I switch from the Neural Differentiation-XF medium to Neural Maintenance Medium-XF?
Please switch to the Maintenance Medium-XF after the suggested period of Neural differentiation medium-XF treatment (see page 12 and 15 of the Human iPSC-Derived Neural Stem Cell Master Protocol below).
A sheet of neurons is peeling from the corners , is this normal and can I stop it?
Please make sure you change medium gently and avoid adding the medium from one side of the wells throughout the 5-6 weeks of culture. If the cells start to peel from the corners, it can be repaired by adding Surebond (ax0041) into your standard feeding media. Usually, we use 120 uL Surebond in 12 mL medium for a few days until the layer re-attach. This can be applied no matter what coating has been used.
When using the XF medium system for iPSC-derived NSCs, what seeding densities should I use?
Please follow the links below: Sync. differentiation: https://www.axolbio.com/web/binary/saveas?model=ir.attachment&field=datas&filename_field=name&id=77214&t=1556543155516 Spontaneous differentiation: https://www.axolbio.com/web/binary/saveas?model=ir.attachment&field=datas&filename_field=name&id=77213&t=1556543179728
Can I use other coating reagents such as Poly-D-Lysine?
We do not recommend using coating reagents whcih we have not tested with our cells. They might result in poor quality cultures and low adherence. Our SureBond, SureBond-XF and SureBond+ReadySet have been optimized to complement our cells and provide consistent results.
Can the iPSC-derived NSCs be expanded and frozen down again?
We do not recommend re-freeze the NSCs. Axol cannot guarantee the viability of the iPSC-derived NSCs and functionality of the neurons derived after re-freezing.
How long can the iPSC-derived NSCs be expanded for?
A long expansion period will increase the number of glial cells in your final population. We recommend conducting fewer passaging steps (< 3 passages) over a shorter period of time in order to reduce the glial cell population.
What is the composition of pure neuronal population in terms of cell types?
It is possible to achieve a 90% pure population of cerebral cortical neurons after terminal differentiation using Neural Differentiation-XF Medium (System B). Repeated expansion of the NSCs will increase the glial population and conversely decrease the neuronal population.
What is the ratio of cortical deep layer neurons (Tbr1 & Ctip2 positive) and upper layer neurons (CUX1 & BRN2 positive) after Axol iPSC-derived NSC differentiation?
The ratio of deep to upper layer neurons will change with the number of days in culture. After 2 weeks in Neural Maintenance-XF Medium, approx. 60% of neurons express deep layer markers but this will decrease with length of time in culture. We would recommend spontaneous differentiation for over 40 days to see a large percentage of upper layer neurons.
When would I expect to see synaptic activity?
At day 21, spontaneous synaptic activities should be detected, and day 35 synchronised burst firing should occur.
Will I be able to detect long-term potentiation and long-term depression?
Yes, Axol iPSC-Derived Cortical Neurons when co-cultured with astrocytes have been shown to respond to high frequency stimulation resulting in a change in spike frequency presenting as a depression of potentiation of network transmission.
What markers are commonly used to detect the Cortical NSCs?
We typically use PAX6, SOX2, Nestin, FOXG1, OTX, ASPM, N-cadherin and Ki67 to identify NSCs.
What markers are commonly used to detect neurons?
NeuN, TBR1, TUJ1, MAP2, GAD67, VGLUT1, Synaptophysin, CTIP2, CUX1 and BRN2 can be used to identify cerebral cortical neurons.
Technical Resources
References
- Thaler A, Ash E, Gan-Or Z et al. The LRRK2 G2019S mutation as the cause of Parkinson's disease in Ashkenazi Jews. Journal of Neural Transmission (2009)
- West AB, Moore DJ, Biskup S et al. Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity. Proc Natl Acad Sci USA (2005)
- Di Fonzo A, Tassorelli C, De Mari M et al: Comprehensive analysis of the LRRK2 gene in sixty families with Parkinson's disease. Eur J Hum Genet (2006)