What general guidelines can you recommend for setting up an assay for iCell DopaNeurons?

While we are not able to evaluate the performance of iCell® DopaNeurons in every assay or functional endpoint, we can provide general guidelines as a basis for you to optimize an assay to meet your needs.

General Protocol Recommendations:

  • Assay ECM: Fresh PLO/laminin double coat
  • Assay density: 160,000 - 230,000 cells/cm2 (51,200 - 73,600 cells/well in a 96-well plate)
  • Assay media: Complete iCell DopaNeurons Maintenance Medium
  • Assay timing: day 7 to 21 post-thaw

Additional Considerations:

  • Remove PLO. It is mportant to remove PLO completely before adding laminin, as PLO is very toxic to cells.
  • Residual laminin is okay. Laminin does not need to be completely removed before plating cells.
  • Do not allow wells to dry out. Laminin coating in wells should NOT be dry before seeding DNC.
  • Remove cryopreservant. Use the recommendations in our User’s Guide to thaw and plate iCell DopaNeurons, paying attention to the resuspension and centrifugation steps. These steps are included to help remove trace amounts of cryopreservant, which can adversely affect plating.
  • Medium changes. Due to the electrical activity of the cells, it is important to perform a 75 - 100% medium change on day 2 or 3 post-thaw and every 2 - 3 days thereafter. Allow cells to recover for at least 48 hours after a medium change before assaying.
  • Network formation. Network formation begins in the first 24 hours and is further established in the first 48 hours.

Alternate ECM. iCell DopaNeurons are not amenable to other ECMs. Cells will not adhere to the culture vessel if a polymer base coat is not used and outgrowth morphology will differ (e.g., shorter outgrowths, thicker near the soma) if no laminin coat is present to provide the amino acid necessary for cell adhesion.  See IDENTIFIER 00000180A for additional information.

:Reference

  1. iCell DopaNeurons User's Guide.