What do you recommend for patch clamping iCell DopaNeurons?

We do not have a fully optimized application protocol for culturing iCell® DopaNeurons intended for manual patch clamp and the various patch clamp rigs. Instead, we offer the following starting point recommendations to adjust as needed for your specific setup.

General Protocol Recommendations:

  • Assay ECM: Precoated PDL/laminin 12-mm glass coverslips; uncoated 15-mm plastic or glass coverslips (coat with appropriate ECMs). Note that our cells have shown better plating success on plastic coverslips rather than glass ones. For more information, see Solution 00000251A.
  • Assay density: 270,000 cells/cm2 (~513,000 cells/well in a 24-well plate) using a minimum seeding density of 160,000 cells/cm2.
  • Assay media: Complete iCell DopaNeurons Medium; antibiotic can be used.
  • Assay timing: Day 7 - 14 post-thaw (may go to 8 - 10 weeks post-thaw).

Additional Considerations:

  • Ideal cells: The ideal cells to patch are large and multipolar.
  • Seals: Create a seal near the axon hillock where the soma meets a neurite, which will be the most electrophysiologically active portion of the cell. If necessary, apply very weak negative pressure to form a tight seal.
  • Resistance requirement: Discard the cell if whole cell resistance is >10MΩ

Reference:

  1. Poster Presentation: DeLaura et al. Development of hIPSC derived midbrain dopaminergic neurons for disease modeling