What recommendations do you have for transfecting iCell DopaNeurons?

Our Application Note highlights how iCell products are compatible with a variety of commercially available transfection reagents, thus enabling the regulation of specific target genes for numerous applications in drug discovery research. Our Application Protocol describes how to deliver plasmid DNA into iCell® DopaNeurons using the ViaFect Transfection Reagent (Promega). We have tried other transfection reagents with less success compared to Viafect, including success with BacMam delivery methods (see IDENTIFIER 00000221A).

General Protocol Recommendation for a 96-well plate:

  • Assay ECM: Poly-L-ornithine (PLO) / laminin coating according to User's Guide; also can use pre-coated poly-D-lysine (PDL) plates for the first coat and manually apply a second coat (or spike cell suspension) with laminin

Assay timing:

  • EARLY: Transfect on day 4 post-thaw and analyze on day 7 post-thaw (72 hours post-transfection)
  • LATE: Transfect on day 21 post-thaw and analyze on day 24 post-thaw (72 hours post-transfection)
  • Assay density: 64,000 cells/well (96-well plate)
  • Assay media: Complete Maintenance Medium (cells are maintained in the regular culture medium during transfection); use Opti-MEM to dilute the transfection reagent
  • Reagent recommendations: Optimal reagent conditions use 4 μl ViaFect reagent to 1 μg total DNA diluted in 100 μl Opti-MEM
  • Transfection mixture preparation:
    1. Add 1 μg DNA to 100 μl Opti-MEM
    2. Pipette 3 – 5 times to mix
    3. Add 4 μl ViaFect to DNA/Opti-MEM mixture
    4. Pipette 3 – 5 times to mix
    5. Wait 5 – 10 minutes
  • Transfection mixture addition to cells (in media):
    1. Add 10 μl/well (aim for middle)
    2. Move in T-motion (or orbital rocker) 1 – 2 minutes
    3. Incubate at 37°C
    4. Perform 100% media change after 24 hours to remove transfection complex

Expected results:

  • GFP expression for ViaFect observed at 24 hours, with a significant increase at 72 hours post-transfection; cells can express GFP for several weeks since iCell DopaNeurons are post-mitotic
  • Flow cytometry shows low toxicity and ~ 50% efficiency for the EARLY time point and ~25% efficiency for the LATE time point: Efficiency was about 25% (live cells) for 10 μl/well for 96-well plate quantified by flow cytometry

References:

  1. iCell DopaNeurons User's Guide
  2. Application Note: Applying Transfection Technologies to Create Novel Screening Models
  3. Application Protocol: Using Liposome-mediated Transfection for Gene Delivery
  4. Webinar Presentation (CDI/Promega):Efficiently Build Relevant In Vitro Models Using Human Stem Cell-Derived Tissue Cells, High Performance Transfection and Novel Multiplexed Reporter Techniques
  5. Anson, 2015: Building Richer Assays: iPSC-Derived Tissue Cells Are a Powerful Addition to the Biologist’s Tool Box