TITLE: Efficient transfection protocol of rainbow trout gills epithelial (RTgill-W1) cell line through nucleofection system.
AUTHORS: Sebastián Escobar-Aguirre, Amanda Escorza, Matias Escobar-Aguirre

[DESCRIPTION]
In this protocol, we optimize the experimental condition to express plasmid DNA in salmonid fish rainbow trout gills epithelial (RTgill-W1), using a nucleofection system. The principle of nucleofection is a combination of electrical parameters that ensures efficient DNA delivery into the nucleus, combined with low toxicity and high cell viability.  Using the Neon Transfection System (Invitrogen‱ MPK5000; code: 10431915) and the Invitrogen‱ Neon‱ Transfection System Kit 10 μl (brand: Invitrogen‱ MPK1096; code: 10124334) we were able to express over 60% of GFP in RTgill W 1 line cells. These results were achieved after trying different optimization steps voltage (ranges between 850 - 1600 volts), pulse exposure (times 10 30 ms), and number of pulses, being the condition 1600 volts, 20 ms 1 pulse, and 1 μg μL of plasmid the most effective compared to the other conditions, presenting a higher percentage of GFP expression and lower mortality rate.


[BEFORE_START]
Cells were grown as a monolayer at 20 °C without CO 2 in Leibovitz L 15 medium (Cityva, USA) supplemented with 10 % heat-inactivated fetal bovine serum (FBS) (Biological Industries, Israel) and maintained at 1 x antibiotics solution ().
The culture must be at 70 - 80% confluency to ensure the exponential growth phase of the cells.

The electroporations will be using 10 µL tips and dispensed in 500 µL of fresh media in a 48- well plate.


[STEPS]
SECTION: Cell preparation
1. Before electroporation, cells should be between about 70 - 90% confluent (i.e 6x10e6 cells/ml in a 175 cm2 flask).
SECTION: Cell preparation
3. Neutralize cells with growth medium 9 mL into the flask.
SECTION: Cell preparation
2. Wash cells monolayer 2  times with  5 mLand 1 mL
SECTION: Electroporation
14. Apply 1600 volts for 20 min and 1 pulse

SECTION: Electroporation
13. Aspirate the cell/plasmidial DNA mix with the Neon tip. BUBBLES INSIDE THE TIP SHOULD BE AVOIDED AS THESE AFFECT THE VOLTAGE TRANSFER.
SECTION: Cell preparation
8. Remove all PBS and resuspend the cells in 10 µL of Neon R buffer.
SECTION: Cell preparation
9. Prepare a 48-well plate with500 µL  where the cells will be seeded post-electroporation.

SECTION: NEON System Preparation
10. Prepare the Neon‱ electroporation tubes with 3 mL and place it inside the Neon Pipette Station.
SECTION: NEON System Preparation
11. Add cells to the tube containing 1 ug/uL and gently mix. This plasmid encodes GFP (green fluorescent protein) as a reporter gene.
SECTION: NEON System Preparation
12. Insert the Neon tip into the Neon pipette (it is necessary to reach the second stop to open the clamp)
SECTION: Electroporation
13.1. Insert the Neon pipette together with the Neon tip into the pipette station vertically until a click is heard
SECTION: Electroporation
15. Carefully remove the Neon pipette from the station and immediately transfer the cells to a 48-well plate with medium WITHOUT ANTIBIOTIC  and gently mix.
SECTION: Determination of transfection efficiency
17. Quantify/Estimate the number of live cells after 72 hours post electroporation.
SECTION: Determination of transfection efficiency
18. Determined transfection efficiency by measuring GFP fluorescence  using the Cytation 5 platform from Agilent Technologies (excitation 469 emission 525).
It is recommended to use Hoechst staining (excitation 377 emission 447) to evaluate cell viability



 





 



SECTION: Cell preparation
5. From the total cell number (step 4), split 105 cells/well (48-well plates) in a new tube (i.e 4 reactions = 4 wells, we would have 4×105 cells).
SECTION: Cell preparation
4. Take an aliquot of trypsinized cell suspension, and register the viable total cell number by trypan blue 1:1 or similar.
SECTION: Electroporation
16. Culture in monolayer at 20 °C without CO2 in Leibovitz L 15 medium supplemented with 10% fetal bovine serum without antibiotic for 24 hours. Then change to fresh culture medium.
SECTION: Cell preparation
6.  Centrifuge it 500 x g, 5 min, 4 °C to pellet the cells.
SECTION: Cell preparation
7.  Remove the supernatant and wash with5 mL and repit step 6.