TITLE: Transfection for Recombinant Antibodies
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
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.

[GUIDELINES]
General Considerations:
For antibody-expressing plasmids containing the SV40 origin of replication use a HEK293 line stably expressing the SV40 large T antigen. 

For antibody-expressing plasmids containing the Epstein-Barr virus origin of replication use a HEK293 line stable expressing Epstein Barr nuclear antigen (EBNA). 

Workflow Timeline
Day 1: Seed cells
Day 2: Transfect cells
Day 3-6: Feed cells
Day 7: Harvest antibody

Tips and Troubleshooting:
We recommend wiping down all pipettes and equipment with 10% bleach prior to use. 

[STEPS]
SECTION: Seeding cells
1. One day prior to transfecting, seed a 108 mL of culture of cells at a density of 0.9*10^6 cells/mL in a 500 mL vented flask.
 


SECTION: Seeding cells
2. Check cell density and viability:


SECTION: Seeding cells
3. Transfer 108 mL of BCD TFX media into each of a 500 mL vented flask.

SECTION: Seeding cells
4. Cap the flask and equilibrate media in a 37 °C, 5% CO2 incubator for 60 min.

SECTION: Seeding cells
5. Transfer the required volume of cells to seed each flask with 108 mL at 0.9*10^6 cells/mL to centrifuge bottles.

SECTION: Seeding cells
6. Centrifuge to pellet cells, 100 x g, 10 min 

SECTION: Seeding cells
7. Aspirate supernatant.
SECTION: Seeding cells
8. Resuspend pellets in 10 mL of the pre-equilibrated media and transfer back into the flask, with total volume 108 mL .

SECTION: Seeding cells
9. Cap the flask.
SECTION: Seeding cells
10. Incubate in a 37 °C, 5% CO2 incubator on a shaking platform set to  .

SECTION: Transfection
11. Place the flask containing HEK293 cells seeded the previous afternoon in the BSC.
 

SECTION: Transfection
12. Using a 5 mL pipette, transfer 0.5 mL of cell suspension into a clean microcentrifuge tube.
 


SECTION: Transfection
13. Transfer 10 µL of trypan blue into a clean microcentrifuge tube.

SECTION: Transfection
14. Vortex the cell suspension.
SECTION: Transfection
15. Transfer 10 µL of cell suspension into the microfuge tube containing the trypan blue.

SECTION: Transfection
16. Pipette 10 times to mix.
SECTION: Transfection
17. Load 10 µL of the cell suspension/trypan blue mix into one chamber of a cell counting chamber slide.

SECTION: Transfection
18. Load the cell counting chamber on an automated cell counter and measure the live cell density and viability of the culture.
 

SECTION: Transfection
19. Transfect the flask containing 108 mL cells as follows:
SECTION: Transfection
19.1. Transfer 6 mL of BCD TFX into each of two 50 mL tubes.

SECTION: Transfection
19.2. Cap the tubes and incubate for 60 min in the 37 °C bead bath to warm.

SECTION: Transfection
19.3. Transfer the PEI-MAX and recombinant antibody plasmid DNA sample to the BSC and incubate for 60 min at Room temperature.

SECTION: Transfection
19.4. After the BCD TFX has warmed in the bead bath, transfer the tubes to the BSC.
SECTION: Transfection
19.5. Add 180 µg of recombinant antibody plasmid DNA to one tube of 6 mL BCD TFX.

SECTION: Transfection
19.6. Cap the tube and vortex for 5 seconds to mix.

SECTION: Transfection
19.7. Add 450 µg of 1 mg/mL PEI-MAX to the second tube of 6 mL BCD TFX. (For a 1 mg/mL stock solution of PEI-MAX, 450 µg = 450 µL)


 



SECTION: Transfection
19.8. Cap the tube and vortex for 5 seconds to mix.

SECTION: Transfection
19.9. Add the diluted PEI-MAX to the diluted DNA.
SECTION: Transfection
19.10. Cap the tube and vortex with three 1-second pulses.
SECTION: Transfection
19.11. Incubate for 3 min at Room temperature.

SECTION: Transfection
19.12. Transfer the 500 mL flask of HEK293 cells to the BSC.
SECTION: Transfection
19.13. Add 12 mL of transfection mix to the flask dropwise.

SECTION: Transfection
19.14. Cap the flasks and swirl 5-10 times to mix.
SECTION: Transfection
20. Return the flask to the incubator.
37 °C 

SECTION: BCD feed and valproic acid supplementation
21. During the 24-144 h post transfection, supplement the flask with 4% BCD feed.
 


SECTION: BCD feed and valproic acid supplementation
22. At 72-96 h post transfection, supplement the flask with 3.75 millimolar (mM) valproic acid.

 

SECTION: Harvesting antibody
23. At 168 hours (1 week) post-transfection, harvest the antibody:
SECTION: Harvesting antibody
24. Transfer the HEK293 cells and media to 50 mL conical tubes.
SECTION: Harvesting antibody
25. Centrifuge to pellet the cells, 3100 x g, 15 min 
SECTION: Harvesting antibody
26. Filter the supernatant through a 0.45 µm PES filter.
SECTION: Harvesting antibody
27. Add 1X protease inhibitor cocktail to the supernatant.
SECTION: Harvesting antibody
28. The supernatant containing the recombinant antibody can be used as-is or the recombinant antibody can be purified from the supernatant. 
 

SECTION: Harvesting antibody
29. Store the supernatant at 4 °C until ready to use.

SECTION: Seeding cells
2.1. Transfer the flask of HEK cells in culture into the biosafety cabinet (BSC).

 

SECTION: Seeding cells
1.1. 
SECTION: Seeding cells
2.2. Transfer 10 µL of trypan blue into a clean microcentrifuge tube.
SECTION: Seeding cells
2.3. Vortex the cell suspension.
SECTION: Seeding cells
2.4. Transfer 10 µL of cell suspension into the microfuge tube containing the trypan blue.
SECTION: Seeding cells
2.5. Pipette 10 times to mix.
SECTION: Seeding cells
2.6. Load 10 µL of the cell suspension/trypan blue mix into one chamber of a cell counting chamber.
SECTION: Seeding cells
2.7. Load the cell counting chamber on an automated cell counter and measure the live cell density and viability of the culture.
SECTION: Seeding cells
2.8. Determine the volume of cell suspension required to seed 108 mL of media at a final density of 0.9*10^6 cells/mL.