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CHO Transfection with LovTAP only
Protocol: Transfection of CHO cells
This is the transfection protocol used at the LBTC lab for CHO DG44 cells. The transfection reagent is PEI ([http://en.wikipedia.org/wiki/Polyethylenimine polyethylenimine]).
Please use the provided Excel sheet to calculate the volume of plasmid you should add to the cells. Replace every value in red by your own, then print the sheet out and follow the provided protocol.
1. Passage seed 1 day prior to transfection.
2. Prepare tubes (yellow caps with holes) by addition of the calculated amount of DNA.
3. Centrifuge the necessary volume of seed (after a PCV measurement), remove conditioned medium with the pump (use a 2 ml serological pipet with a broken neck) and resuspend (first in 10 ml) in necessary volume of fresh medium to achieve the required cell density (3 mio/ml).
4. Add 5 mL of the cell suspension to the tube with DNA and mix orbitally.
5. Add the PEI (45 µl) to the Cell+DNA mixture as soon as possible, flick 3 times.
6. Place in the incubator at 37°C.
We trasfected a batch of CHO cells with LovTAP only. We made two tubes with 100% LovTAP and one with 95% LovTAP and 5% pOri-eGFP (this is a control plasmid with a very high constitutive expression of GFP, it is used to check if the transfection has worked, with the Guava).
Guava Measurements on HEK cells transfected with pHY42 and a GFP readout
Team:EPF-Lausanne/Protocol/Guava-Flowcytometry
A routine measurement of green fluorescence has been performed on the HEK cells transfected with melanopsin. We didn't see a big difference of GFP expression between transfected and non-transfected cells. However, we can notice that the cells that have been under the Arduino blue light for a while have a different shape and less viability.
