Transfection for Recombinant Antibodies
Addgene The Nonprofit Plasmid Repository
Disclaimer
We have listed the specific equipment, reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other vendors should produce similar outcomes when using these protocols. However, please be aware that your protocol may need to be adjusted to accommodate slight differences between products. Addgene does not endorse or recommend specific products or equipment. Inclusion of this information is solely for transparency intended to support reproducibility in science.
Abstract
This protocol describes how to transfect suspension HEK293 cells with recombinant antibody plasmids using Polyethylenimine Max as a transfection reagent. After transfection and expression, the recombinant antibody can be purified for use in a variety of applications.
See the original protocol on Addgene's website: https://www.addgene.org/protocols/transfection-for-recombinant-antibodies/
Before start
See the Materials section for preparation of necessary stock solutions.
Warm the DNA and working stock of PEI-MAX to room temperature before use, and warm the BalanCD HEK293 transfection media (BCD TFX) to 37 °C before use.
Steps
Seeding cells
One day prior to transfecting, seed a 108mL of culture of cells at a density of 0.9*10^6 in a 500 mL vented flask.
Check cell density and viability:
Transfer the flask of HEK cells in culture into the biosafety cabinet (BSC).
Transfer 10µL of trypan blue into a clean microcentrifuge tube.
Vortex the cell suspension.
Transfer 10µL of cell suspension into the microfuge tube containing the trypan blue.
Pipette 10 times to mix.
Load 10µL of the cell suspension/trypan blue mix into one chamber of a cell counting chamber.
Load the cell counting chamber on an automated cell counter and measure the live cell density and viability of the culture.
Determine the volume of cell suspension required to seed 108mL of media at a final density of 0.9*10^6.
Transfer 108mL of BCD TFX media into each of a 500 mL vented flask.
Cap the flask and equilibrate media in a 37°C, 5% CO2 incubator for 1h 0m 0s.
Transfer the required volume of cells to seed each flask with 108mL at 0.9*10^6 to centrifuge bottles.
Centrifuge to pellet cells, 100x g
Aspirate supernatant.
Resuspend pellets in 10mL of the pre-equilibrated media and transfer back into the flask, with total volume 108mL .
Cap the flask.
Incubate in a 37°C, 5% CO2 incubator on a shaking platform set to 120rpm.
Transfection
Place the flask containing HEK293 cells seeded the previous afternoon in the BSC.
Using a 5 mL pipette, transfer 0.5mL of cell suspension into a clean microcentrifuge tube.
Transfer 10µL of trypan blue into a clean microcentrifuge tube.
Vortex the cell suspension.
Transfer 10µL of cell suspension into the microfuge tube containing the trypan blue.
Pipette 10 times to mix.
Load 10µL of the cell suspension/trypan blue mix into one chamber of a cell counting chamber slide.
Load the cell counting chamber on an automated cell counter and measure the live cell density and viability of the culture.
Transfect the flask containing 108mL cells as follows:
Transfer 6mL of BCD TFX into each of two 50 mL tubes.
Cap the tube and vortex with three 1-second pulses.
Incubate for 0h 3m 0s at Room temperature.
Transfer the 500 mL flask of HEK293 cells to the BSC.
Add 12mL of transfection mix to the flask dropwise.
Cap the flasks and swirl 5-10 times to mix.
Cap the tubes and incubate for 1h 0m 0s in the 37°C bead bath to warm.
Transfer the PEI-MAX and recombinant antibody plasmid DNA sample to the BSC and incubate for 1h 0m 0s at Room temperature.
After the BCD TFX has warmed in the bead bath, transfer the tubes to the BSC.
Add 180µg of recombinant antibody plasmid DNA to one tube of 6mL BCD TFX.
Cap the tube and vortex for 5 seconds to mix.
Add 450µg of 1 mg/mL PEI-MAX to the second tube of 6mL BCD TFX. (For a 1 mg/mL stock solution of PEI-MAX, 450 µg = 450 µL)
Cap the tube and vortex for 5 seconds to mix.
Add the diluted PEI-MAX to the diluted DNA.
Return the flask to the incubator.
37°C
BCD feed and valproic acid supplementation
During the 24-144 h post transfection, supplement the flask with 4% BCD feed.
At 72-96 h post transfection, supplement the flask with 3.75millimolar (mM) valproic acid.
Harvesting antibody
At 168 hours (1 week) post-transfection, harvest the antibody:
Transfer the HEK293 cells and media to 50 mL conical tubes.
Centrifuge to pellet the cells, 3100x g
Filter the supernatant through a 0.45 µm PES filter.
Add 1X protease inhibitor cocktail to the supernatant.
The supernatant containing the recombinant antibody can be used as-is or the recombinant antibody can be purified from the supernatant.
Store the supernatant at 4°C until ready to use.
