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Q and A Advanced models of Parkinson’s disease

Advanced models of Parkinson’s disease questions and answers
  1. Often neuronal cultures require the use of plates coated with a substrate such as poly-d-ornithine or poly-l-lysine. Does neural progenitor cell (NPC) culture require a gel coating or substrate?

    ATCC NPC protocols require that the culture dishes be coated with CellMatrix Basement Membrane Gel (ATCC® ACS-3035™)

  2. How many passages can Neural Progenitor Cells Derived from ATCC-DYS0530 Parkinson's Disease (ATCC® ACS-5001™) undergo?

    ATCC NPCs can be proliferated at least 15 PDLs and passaged over 20 times. Some of the results shown in the webinar were using cells from passage 10.

  3. Can you explain why you are getting different neuronal population when you claim your media is dopaminergic differentiation media?

    The majority of the growth factors in our differentiation medium are common to generate all neuronal cell types; the big difference is the concentration needed to make a majority of a specific neuron type. By tweaking the concentration of growth factors we were able to increase the number of dopaminergic neurons relative to other neuron types.

  4. What dissociation reagent is recommended for the passaging of NPCs?

    ACS-5001 NPCs require undiluted Accutase for passaging that differs from the protocol for normal NPCs which uses Accutase diluted 1:1 with DPBS.

  5. You mentioned the addition of CHIR-99021 to the media for differentiating the Parkinson’s disease NPCs into dopaminergic neurons. Where did you get the compound from, and what concentration is it used?

    For differentiating ACS-5001 NPCs to dopaminergic neurons, we must add CHIR-99021 at a final concentration of 5 µM (Stemgent cat. 04-0004-10) to the dopaminergic differentiating media (ATCC® ACS-3004™). This component can be added in an aliquot of dopaminergic media and store it at 4 C for a week.

  6. What is the optimal cell confluence when passaging NPCs?

    Unlike other primary cells, passaging NPCs near 100% confluence doesn’t affect cell performance. In fact, it is highly recommended to passage NPCs when they reach ~95% confluence. Do not split NPCs when they are < 85% confluence, which will inhibit cell proliferation.

  7. Are there passaging recommendations for NPCs during tri-lineage differentiation?

    It is OK to split cells during astrocyte and oligodendrocyte differentiation when NPCs become confluent. However, passaged NPCs will not survive well during dopaminergic neuron differentiation. Over-confluence of NPCs will not affect the outcome of dopaminergic neuron differentiation.