Protocol: Plasmid transfection

Use VersaLive to run plasmid transfection on chip.

Materials

  • Cell medium
  • Transfection mix
  • Mineral oil for cell culture

Outline_protocol.png

Transfection mix

It is advised to find the most suitable transfection mix of DNA and lipid complex concentrations via a screening in 96-well plate.

For our system (plasmid with mCherry fluorescent reporter expressed from the CMV promoter applied on HeLa cells), it was found that the optimal combination contained 300 ng of DNA, 0.5 μL of lipid complex and 10 μL of OptiMEM. 

For the on-chip transfection, the mix was diluted with cell growth medium to a final volume of 50 μL. 

Aliquots of this stock transfection mix were used to perfuse the cells over 2 hours in multi-input mode. By using the multi-input mode, it is also possible to test multiple plasmids on the same chip.

Cell culture on VersaLive

Follow the protocol relative to the cell culture on VersaLive. Calibrate the cell loading to have a cell coverage in the chambers close to 80% by the time of the on-chip transfection. The transfection can start as soon as the cells start to appear flattened (not round).

On-chip transfection

  1. Empty all reservoirs
  2. Add 5 μL of stock transfection mix in reservoirs #1 to #5 (leave ports A and B empty)
  3. Cover the mix with at least 2.5 μL of mineral oil to prevent evaporation
  4. Place the chip in incubator for 2 hours of perfusion
  5. Empty all reservoirs
  6. Add 5 μL of cell medium to all reservoirs (static cell culture)
  7. Add 2.5 μL of mineral oil to each reservoir to prevent the evaporation of the cell medium
  8. Place the chip in incubator for 24 hours
  9. Acquire microscopy data