Electroporation of Recombinant Proteins for in vivo Functional Studies in Cultured Mammalian CellsPreprint   

Abstract

Materials and Reagents

1. 50 ml conical Falcon tubes (Corning, Falcon, catalog number: 430290)
2. 15 ml conical Falcon tubes (Corning, Falcon, catalog number: 352095) 
3. 1.5 ml Eppendorf tubes (Sigma-Aldrich, Eppendorf, catalog number: Z66505-100EA) 
4. Sterile individually packaged 5 ml pipettes (Sigma-Aldrich, catalog number: SIAL1487)
   
5. Sterile individually packaged 10 ml pipettes (Sigma-Aldrich, catalog number: SIAL1488)
6. 24 well imaging plate (Ibidi, catalog number: 82406)
7. Corning 10 cm cell culture dish (Sigma-Aldrich, catalog number: CLS430167)
8. HeLa cells
9. Purified protein of interest
10. Buffer R (included in Neon Transfection Kit, see Equipment section below)
11. FBS (Sigma-Aldrich, catalog number: TMS-013)
12. Penicillin, Streptomycin (GIBCO, catalog number: 15140122) 
13. L-glutamine (GIBCO, catalog number: 25030081)
14. DMEM (Sigma-Aldrich, catalog number: D6429)
15. Phosphate Buffered Saline (PBS) (GE Healthcare, catalog number: SH30013.03) without calcium and magnesium
16. Trypsin EDTA (Sigma-Aldrich, catalog number: T4799)
17. CO₂ independent imaging media (GIBCO, catalog number: 18045054)
18. NaCl (Sigma, catalog number: S9888-1KG)
19. KCl (Sigma, catalog number: P3911-500G)
20. Na₂HPO₄ (Sigma, catalog number: S3264)
21. KH₂PO₄ (Sigma, catalog number: P5655-500G)
22. HCl (Sigma)
23. HeLa Cell growth medium (see Recipes)
24. 1x PBS (see Recipes)
    

Equipment

1. Pipetman P20 pipette (Gilson, catalog number: F123600) 
2. Pipetman P200 pipette (Gilson, catalog number: F123601) 
3. Pipetman P1000 pipette (Gilson, catalog number: F123602) 
4. Pipette boy (Corning, Falcon, catalog number: 357469) 
5. Water bath (Julabo, model: ED (v.2)) 
6. Eppendorf centrifuge 5804 R (Eppendorf, model: 5804 R, catalog number: 805000620)
7. Countess Cell Counter (Thermo-Fisher, catalog number: AMQAX1000) 
8. Neon Transfection System (Invitrogen, catalog number: MPK5000S)
9. Neon Transfection 100 μl Kit (Invitrogen, catalog number: MPK10025, this Kit includes also Buffer R, which can not be purchased separately)
10. Deltavision Elite System (GE Healthcare) 
11. Cell culture incubator with CO₂ supply 
12. Cell culture hood
    

Procedure

Electroporation (EP):

1. Culture the cells on 10 cm cell culture dishes until they reach the desired confluency, e.g., for HeLa cells we use 70-80% confluency.
2. Pre-warm 2.5% trypsin, the PBS and the growth culture medium without antibiotics to 37 °C prior to the experiment.
3. Remove the cell culture medium and wash the cells once with 5 ml of PBS.
4. Detach the cells from the surface by incubating them with 1 ml of 2.5% trypsin for 5 min at 37 °C. 
5. Harvest the trypsinized cells and add to a 50 ml tube and use warm PBS to bring the volume to 50ml. Pellet the cells by centrifuging them at 500 x g for 5 min at 25 °C.
6. Remove supernatant and resuspend the cells in approximately 1 ml of PBS and count the cell number using the cell-counter Countess Cell Counter (Equipment number 7). Cell density should range between 2 and 8 x 10⁶ cells/ml.
7. Transfer the volume of cell suspension needed for a final cell number between 1 and 3 x 10⁶ cells to a 1.5-ml Eppendorf and pellet the cells by centrifuging them at 500 x g for 5 min.
8. Remove the PBS and resuspend the pellet to a final 90 μl of resuspension buffer (buffer R). This suspension is called “EP slurry”. It is important to proceed immediately with the EP, after this step. Cell viability could decrease significantly if the cells remain longer than 2 min in buffer-R. Buffer R is part of the EP kit from Invitrogen and its proprietary composition is unknown to the authors. We suggest to leave cell pellet in PBS and proceed at the resuspension in buffer R and EP one sample at the time.
9. Dilute the recombinant purified protein 1:1 (volume:volume) in buffer R and add 30 µl of this suspension to the 90 µl of EP slurry, for a final volume of 120 µl. Volumes and protein-buffer ratios may be adjusted according to the purpose of the experiment and depending on protein solubility. Final protein concentrations in the respective EP mixtures we tried varies between 5 and 200 µM.
10. Load the EP mixture into a 100 µl Neon Pipette Tip making sure to avoid bubble in the Neon tip while pipetting, as this will cause an electric short-circuit and kill the cells during EP. 
11. Put the Neon pipette into the pipette station and electroporate the cells using the following:
    parameters:
    Voltage: 1,000 V
    Number of pulses: 2
    Time width of pulse: 35 ms 
12. Following the EP, add the EP-mixture to 15 ml of pre-warmed PBS in a 15 ml Falcon tube and centrifuge at 500 x g for 5 mins.
13. Remove the PBS and add 3 to 7 ml of Trypsin to the cell pellet (according to the pellet size) and incubate for 5 min at 37 °C. This step of trypsinization helps to remove non-internalized extracellular protein, and its timing needs to be adjusted on a protein-specific basis.
14. Make up the volume in the Falcon tube to 15 ml using pre-warmed PBS and pellet the cells for 5 min at 500 x g.
15. Remove the PBS and repeat the washing Step 13 two more times.
16. Resuspend the cell pellet in complete Imaging media (without antibiotics) and transfer to a cell-imaging plate.
17. Keep the plate in 37 °C incubator and allow the cells to recover for a minimum of 4 h, until cells display adhesion and morphology state resemblying the pre-electroporation conditions.
18. After recovery, analyse the cells under a fluorescent microscope.

Notes

1. This protocol is intended for the electroporation of a fluorescent protein into HeLa cells and observing the localization using fluorescence microscopy. 
2. The quality of the recombinant protein sample is crucial for the success of this protocol. Samples should be monodispersed (i.e., following a Gel filtration purification step) and possibly pure.
3. The Neon transfection system and the corresponding Neon transduction kits are used for EP, but this procedure is also suitable for other electroporators, such as, for example, the Amaxa electroporator or the NEPA21 electroporator.
4. The volumes of buffers and protein mix described in this protocol refers to electroporations performed using the 100 µl Neon tip. 
5. EP parameters need to be adjusted on a cell type specific basis, depending on the trade-off between efficiency of delivery and viability after EP.
   

Recipes

1. HeLa Cell growth medium
   Supplement DMEM with 10% FBS, penicillin, streptomycin and 2 mM L-glutamine
   Store at 4 °C
2. 1x PBS
   For 10 liters of 1x PBS, resuspend one package of Phosphate Buffered Saline (PBS) powder (GE Healthcare) in a final volume of 10 liters of distilled water
   

Acknowledgments

We thank Melina Schuh and Dean Clift for discussion and comments. We are grateful to all members of the Selenko and Musacchio laboratories for reagents, helpful discussions and comments. A.M. gratefully acknowledges funding by the Max Planck Society and the European Research Council (ERC) Advanced Investigator Grant RECEPIANCE (proposal number: 669686).

Competing interests

All authors have no conflict of interest to declare.

References

1. Alex, A., Piano, V., Polley, S., Stuiver, M., Voss, S., Ciossani, G., Overlack, K., Voss, B., Wohlgemuth, S., Petrovic, A., Wu, Y., Selenko, P., Musacchio, A. and Maffini, S. (2019). Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology. Elife 8: e48287.
2. Inomata, K., Ohno, A., Tochio, H., Isogai, S., Tenno, T., Nakase, I., Takeuchi, T., Futaki, S., Ito, Y., Hiroaki, H. and Shirakawa, M. (2009). High-resolution multi-dimensional NMR spectroscopy of proteins in human cells. Nature 458(7234): 106-109.
3. Komarova, Y., Peloquin, J. and Borisy, G. (2007). Microinjection of protein samples. CSH Protoc 2007: pdb prot4657.
4. Ogino, S., Kubo, S., Umemoto, R., Huang, S., Nishida, N. and Shimada, I. (2009). Observation of NMR signals from proteins introduced into living mammalian cells by reversible membrane permeabilization using a pore-forming toxin, streptolysin O. J Am Chem Soc 131(31): 10834-10835.
5. Teng, K. W., Ishitsuka, Y., Ren, P., Youn, Y., Deng, X., Ge, P., Lee, S. H., Belmont, A. S. and Selvin, P. R. (2016). Labeling proteins inside living cells using external fluorophores for microscopy. Elife 5: 20378.
   

1. Alex, A., Voss, B., Musacchio, A. and Maffini, S. (2020). Electroporation of Recombinant Proteins for in vivo Functional Studies in Cultured Mammalian Cells. Bio-101: e5003. DOI: 10.21769/BioProtoc.5003.
   
2. Alex, A., Piano, V., Polley, S., Stuiver, M., Voss, S., Ciossani, G., Overlack, K., Voss, B., Wohlgemuth, S., Petrovic, A., Wu, Y., Selenko, P., Musacchio, A. and Maffini, S. (2019). Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology. Elife 8: e48287.