Hi all,
Has anyone used the Neurite Outgrowth Staining kit (Thermo fisher A15001) for primary neurons / iPSC derived neurons? If you have experience with this I have some questoins (see below)
- I haven't found publications using this system - can anyone point me to one?
- What density is it recommend to plate the neurons at? Is clumping of cells an issue?
- Positive controls - is it enough just to have whatever the control of your experiment?
- Negative controls - is there a compound / method you would recommend to reduce neurite outgrowth?
Any help at all would be very much appreciated!
Below is the citation for the paper that characterized this kit. They tested immortalized, primary, and iPSC neurons and a number of positive and negative control agents. check Thermo website for assay setup guidance too. I may test this kit in the coming weeks and will let you know my impressions.
to answer your other questions: clumping can be frustrating as it makes neurite measurement very difficult if not impossible. To minimize clumping, good thorough dissociation is critical. Optimize seeding density based on type and quantity of neurons you are testing. usually straightforward but can be tricky With primary neurons.
When at all possible, include both positive and negative controls. For assays or kits like this that are newer, inclusion of both controls is even more critical. Positive controls are plentiful: anti cancer agents (taxol, vincristine, oxaliplatin) and mitotoxins (rotenone, CCCP) have been published on extensively. There are many many others as well. If possible, consider using one related to your experimental condition or test drug.
. Hancock, M., Kopp, L., Kaur, N., & Hanson, B. J. (2015). A Facile Method for Simultaneously Measuring Neuronal Cell Viability and Neurite Outgrowth. Current Chemical Genomics and Translational Medicine, 9, 6–16. http://doi.org/10.2174/2213988501509010006
Hi Peter,
Yes I've seen that paper - if you do end up using this kit (what model are you using?) I would be very interested in hearing your opinion of it! Since the authors of that paper are employed by TF I would be interested in getting an impartial view.
Thanks for your help and advice.
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