Sep 23, 2022
Transfection for Recombinant Antibodies
- 1Addgene
Protocol Citation: Addgene The Nonprofit Plasmid Repository 2022. Transfection for Recombinant Antibodies. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g741oqgwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: March 24, 2022
Last Modified: September 23, 2022
Protocol Integer ID: 59879
Keywords: recombinant antibodies, transfection, HEK293, Polyethylenimine Max (PEI-MAX)
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/
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.
Materials
Equipment:
- Class II, Type A2 Biological Safety Cabinet (Tissue Culture hood)
- 4 °C Refrigerator
- Benchtop centrifuge compatible with 50 mL conical tubes
- 37 °C, 5% CO2 incubator with shaking platform set to 120 rpm
- 37 °C bead bath
- Automated cell counter
- Pipet controller
- 0.5-10 µL single channel pipettor
- 2-20 µL single channel pipettor
- 20-200 µL single channel pipettor
- 0.1-1 mL single channel pipettor
- Vortex
- Stir bar
- Magnetic stir plate
- pH meter
Reagents and Consumables:
- White disposable sleeves, DuPont IC501BWH000100CS
- Kimwipes, VWR 82003-822
- Microcentrifuge tubes, VWR 89126-724
- 50 mL conical tubes, VWR 89039-656
- 500 mL vented flask, 431147
- Pipettes and micropipette tips
- Cell counting chamber slide, Thermo Fisher C10228
- 30 mL Luer-lock syringe, BD BD302832
- 0.2 µm Luer-lock filter, VWR 431229
- 0.22 µm PES filtering system, 1000 mL, VWR 431098
- 0.45 µm PES filtering system, 500 mL, VWR 430770
- Trypan Blue, Thermo Fisher T10282
- 10% Pluronic F-18, Thermo Fisher 24040032
- Polyethylenimine hydrochloride, M.W. 40000 (PEI-MAX), Linear, Transfection Grade, VWR 75800-188
- Valproic acid sodium salt, Sigma Aldrich P4543-10G
- Glutagro, Corning 25-015-CI
- BalanCD HEK293 Media, Irvine Scientific 91165-1L
- BalanCD HEK293 Feed, Irvine Scientific 91166-500ML
- Benzamide, Millipore Sigma 12072
- Antipain, Millipore Sigma 10791
- Leupeptin, Millipore Sigma L2884
- Aprotinin saline solution, Millipore Sigma A6279
- HEK293 cells
- Recombinant antibody plasmid DNA
Reagent Preparation:
100 mM Valproic Acid
- Dissolve 2.88 g valproic acid in 200 mL deionized water.
- Sterilize by passage through a sterile 0.22 μm PES filter.
- Prepare 10 mL aliquots and store the solution at -20 °C .
1 mg/mL PEI-MAX
- Add 1 g of PEI-MAX powder to 900 mL deionized water in a 1 L bottle.
- Add stir bar and stir on a magnetic stir plate.
Note
The powder should dissolve rapidly (with 20 min) but check for the presence of particles still in solution. Continue to stir until all particles have dissolved. This may take several hours.
- Adjust to 7.0 as needed with 10 N sodium hydroxide (NaOH) or 5 N hydrochloric acid (HCl).
Note
Adjust the pH slowly, adding no more than 200 µL of NaOH or 20 µL of HCl at a time.
- Add deionized water to a final volume of 1 L . Check to ensure the pH has not drifted.
- Sterilize the solution by passing the solution through a 0.22 µm PES filter apparatus.
- Prepare aliquots and store at -20 °C until use.
BCD TFX
- 1000 mL BalanCD HEK293 Media
- 10 mL 10% Pluronic-F68
- 40 mL 200 mM Glutagro
- Do not add selective reagents.
- Store at 4 °C until use. We suggest preparing fresh solutions after one month.
BCD Feed
- 500 mL BalanCD HEK293 Feed
- 20 mL 200 mM Glutagro
- Store at 4 °C until use. We suggest preparing fresh solutions after one month.
1000X protease inhibitor cocktail
- 25 mg leupeptin
- 50 mg antipain
- 250 mg benzamidine
- 25 mL of 2 mg/mL aprotinin
- Mix well and sterilize through a 0.2 µm PES filter.
- Aliquot and freeze upright at -20 °C until use.
Safety warnings
HEK293 cells are considered biosafety level 2. Please ensure that you are in compliance with your institution’s biosafety regulations.
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.
Seeding cells
Seeding cells
One day prior to transfecting, seed a 108 mL of culture of cells at a density of 0.9*10^6 Mass Percent in a 500 mL vented flask.
Note
Do not use cells that are over 30 passages.
Check cell density and viability:
Transfer the flask of HEK cells in culture into the biosafety cabinet (BSC).
Note
Do not get the filter cap wet.
Transfer 10 µL of trypan blue into a clean microcentrifuge tube.
Vortex the cell suspension.
Transfer 10 µL of cell suspension into the microfuge tube containing the trypan blue.
Pipette 10 times to mix.
Load 10 µL 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 108 mL of media at a final density of 0.9*10^6 Mass Percent .
Note
Refer to the following example for calculating the dilution:
If live cell density = 3 * 106 cells/mL:
C1 * V1 = C2 * V2
3 * 106 cells/mL * V1 = (0.9 * 106 cells/mL) * 108 mL
V1 = 32.4 mL
You would dilute 32.4 mL of the initial 3 * 106 cells/mL culture to a final volume of 108 mL in a 500 mL vented flask.
Transfer 108 mL 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 01:00:00 .
1h
Transfer the required volume of cells to seed each flask with 108 mL at 0.9*10^6 Mass Percent to centrifuge bottles.
Centrifuge to pellet cells, 100 x g, 00:10:00
10m
Aspirate supernatant.
Resuspend pellets in 10 mL of the pre-equilibrated media and transfer back into the flask, with total volume 108 mL .
Cap the flask.
Incubate in a 37 °C , 5% CO2 incubator on a shaking platform set to 120 rpm .
Transfection
Transfection
Place the flask containing HEK293 cells seeded the previous afternoon in the BSC.
Note
Do not get the filter cap wet.
Using a 5 mL pipette, transfer 0.5 mL of cell suspension into a clean microcentrifuge tube.
Note
Cells settle quickly and need to be resuspended before sampling. Gently swirl the flask 5-10 times before sampling.
Transfer 10 µL of trypan blue into a clean microcentrifuge tube.
Vortex the cell suspension.
Transfer 10 µL of cell suspension into the microfuge tube containing the trypan blue.
Pipette 10 times to mix.
Load 10 µL 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.
Note
Culture should be between 1.5 - 2 * 106 cells/mL with >95% viability to proceed with transfection.
Transfect the flask containing 108 mL cells as follows:
Transfer 6 mL of BCD TFX into each of two 50 mL tubes.
Cap the tubes and incubate for 01:00:00 in the 37 °C bead bath to warm.
1h
Transfer the PEI-MAX and recombinant antibody plasmid DNA sample to the BSC and incubate for 01:00:00 at Room temperature .
1h
After the BCD TFX has warmed in the bead bath, transfer the tubes to the BSC.
Add 180 µg of recombinant antibody plasmid DNA to one tube of 6 mL BCD TFX.
Cap the tube and vortex for 5 seconds to mix.
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)
Note
The optimal ratio of DNA:PEI may vary significantly and should be empirically determined for your sample. Typical ratios may range from 1:1 to 1:6.
Cap the tube and vortex for 5 seconds to mix.
Add the diluted PEI-MAX to the diluted DNA.
Cap the tube and vortex with three 1-second pulses.
Incubate for 00:03:00 at Room temperature .
3m
Transfer the 500 mL flask of HEK293 cells to the BSC.
Add 12 mL of transfection mix to the flask dropwise.
Cap the flasks and swirl 5-10 times to mix.
Return the flask to the incubator.
37 °C
BCD feed and valproic acid supplementation
BCD feed and valproic acid supplementation
1w
1w
During the 24-144 h post transfection, supplement the flask with 4% BCD feed.
Note
The feed can be repeated up to 4 times for a total of 16% of the culture volume.
At 72-96 h post transfection, supplement the flask with 3.75 millimolar (mM) valproic acid.
Note
Example feeding strategy:
Thursday: Transfect cells.
Friday (24 h post transfection): Add 4% BCD feed.
Monday (96 h post transfection): Add valproic acid to 3.75 mM, add 4% BCD Feed.
Tuesday (120 h post transfection): Add 4% BCD feed.
Wednesday (144 h post transfection): Add 4% BCD feed.
Thursday (168 h post transfection): Harvest antibody
Harvesting antibody
Harvesting antibody
15m
15m
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, 3100 x g, 00:15:00
15m
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.
Note
Store the supernatant at 4 °C until ready to use.