What do you recommend for using iCell DopaNeurons on FLIPR system?
CDI has developed an Application Protocol that describes how to handle iCell® DopaNeurons for use on the Tetra High Throughput Cellular Screening System (FLIPR Tetra system, Molecular Devices) and provides basic instructions for compound treatment, data acquisition, and analysis. In the protocol we demonstrate that the iCell DopaNeurons can be cultured on 96- or 384-well plates where they form a stable, electrically active network amenable to measuring drug-induced perturbations. These perturbations can be assayed in a high-throughput manner by monitoring calcium transients on FLIPR for in vitro screening of compound efficacy and toxicity in human neuronal cells.
The ideal time to run FLIPR assays with the most consistent and uniform results is after 15 days of culturing iCell DopaNeurons, where screening can be performed on the same day as calcium dye addition to reduce potential for toxicity. Because assay timing is critical and a controlled temperature environment is important, be sure to adhere to the suggested workflow provided in our Application Protocol and pay attention to temperature, always returning plates to 37°C incubator during incubation steps and keeping run times short. The FLIPR systems from Molecular Devices and Hamamatsu both have adequate temperature controls.
General Protocol Recommendations:
- Assay timing: day 15 post-plating
- Assay density (direct plating): 20,000 plated cells in 25 μl per well in 384-well plate (800,000 cells/ml); 50,000 plated cells in 100 μl per well in 96-well plate (500,000 cells/ml); a higher than normal density may be better based on preliminary observations.
- Assay media: Complete iDopaNeuron Maintenance Medium (needs both Medium Supplement and Nervous System Supplement to complete media)
- Assay ECM: PLO (first coat); laminin (second coat); the choice of ECM is critical and PLO/laminin double-coated plates are preferred over PDL pre-coated plates