How do you recommend culturing iCell DopaNeurons for a dopamine release assay?

We have not optimized an application protocol for the use of iCell® DopaNeurons in a dopamine release assay. However, we do have recommendations for running this assay with an ELISA readout.

General Protocol Recommendations:

  • Assay ECM: Fresh poly-L-ornithine (PLO) / laminin double coat
  • Assay density: 3.35 million cells/well in a 12-well plate (~881,500 cells/cm2); Note that using a lower density (e.g., 1.25 million cells/well in 12-well plate) results in lower dopamine levels detectable only at a late time in culture (day 20 to 60 post-thaw).
  • Assay media: HBSS (with 56 mM KCI (stimulated, +KCl) or without KCl (unstimulated, –KCl))
  • Preservatives: 1 mM EDTA and 4 mM sodium metabisulfite
  • Assay timing: Day 14 post-thaw

Protocol Summary:

  1. Prepare PLO/laminin coated culture plates.
  2. Thaw iCell DopaNeurons according to the User's Guide.
  3. Plate 3.35 million cells/well of a 12-well plate.
  4. Maintain cultures for 14 days according to the User's Guide.
  5. On day 14 post-thaw, rinse plate with 2 ml HBSS.
  6. Aspirate and add 300 µl HBSS +/– KCl.
  7. Incubate for 30 minutes at 37°C.
  8. Collect HBSS.
  9. Add fresh Maintenance Medium warmed to room temperature. Important: We do NOT recommend using a DMEM-based medium, which has been found to inhibit ELISA kit measurements to values near or below the zero control.
  10. If maintaining cells in culture for additional analysis:
    • Add EDTA from a 1000x stock solution to a final concentration of 1 mM.
    • Add sodium metabisulfite from a 1000x stock solution to a final concentration of 4 mM.
  11. Immediately run ELISA assay or store supernatant with preservatives in –80°C for up to 2 weeks. Note that supernatant stored frozen at –80°C can result in lower dopamine values but with similar trends.

Reference:

  1. iCell DopaNeurons User’s Guide