How do you recommend using iCell DopaNeurons on FDSS?

Currently we do not have an application protocol that describes how to handle iCell® DopaNeurons for use on the FDSS platform. We do have a Poster showing spontaneous Ca2+ oscillations in iCell DopaNeurons and can provide basic instructions and protocol recommendations for using iCell DopaNeurons on the FDSS.

General Protocol Recommendations:

  • Assay timing: 14 days
  • Assay density: 200,000 cells/cm2 or higher (≥ 64,000 cells/well) in 96-well plate (Note that at a higher density, the signal amplitude is bigger.)
  • Assay media: Complete BrainPhys Medium from thaw or Complete DopaNeurons Maintenance Medium switched to Complete BrainPhys Medium on day 12 (2 days prior to assay)
  • Assay ECM: Poly-D-lysine (PDL) pre-coated or poly-L-ornithine (PLO), with second coat of laminin
  • Assay solution: Modified Tyrode’s Solution without Mg2+, pH 7.4
Component Concentration (mM)
NaCl 135
KCl 5
D-glucose 10
HEPES 10
CaCl2 2.5
  • Culture vessel: Corning 356640 or Greiner 655946

Dye Reagent Dilutions:

Part Stock Conc. 100% Medium Exchange-1X 50% Medium Exchange-2X
Fluo-4 AM 1 mM in DMSO 1 μl/ml media (1 μM) 2 μl/ml
Probenecid 250 mM in water 4 μl/ml media (1 mM) 8 μl/ml
Pluronic F-127 10% in water 2 μl/ml media (0.02%) 4 μl/ml

Reference:

  1. Poster Presentation: Calcium Handling Assays with Human iPSC-derived Cell Types on the Hamamatsu FDSS/μCell