ein “transfection” reagents 15, calcium phosphate precipitations 16, and electroporation 17-20. The introduced molecules range from nucleic acids including DNA, RNA, & RNAi species to proteins, cell-impermeable dyes, and antibodies for intracellular targets 21,22. Some methods have limitations including the type of macromolecule that can be introduced, and particular downstream analyses may be limited due to high cellular toxicity, damaging mechanisms of action, low efficacy, or introduction efficiency. Transfection, an often-used method for expressing bacterial genes in mammalian cells, also suffers the limitation that some relevant host cell types, such as macrophages and primary cells, are particularly resistant towards transfection. Beyond this, it is difficult to control the levels of bacterial protein produced upon introduction of foreign DNA.
Much work has established electroporation of nucleic acids into both bacterial and mammalian cells as a common laboratory technique; however, there is ongoing research into the best methods for delivering proteins into cells under physiological conditions. Reports on protein transfection are promising, but require expensive reagents and optimization. The desire to introduce potentially toxic bacterial effectors into a wide variety of cell targets with minimal cost led us to consider electroporation as a method for studying these proteins in vivo.
Protein electroporation 23-25 is a method to introduce proteins into living cells via electropermeabilization, also known as electro-transfection or electro-injection 26. This technique uses high-intensity electrical pulses to create pores in cell membranes. These reversible pores allow macromolecules that are normally excluded from intracellular space to enter the cell. Upon removal of the external electric field, the membrane can reseal, allowing the cell to retain molecules that passed through the pores 27,28.
A standard electroporator was used in this study to consistently introduce bacterial effectors into both mouse macrophage-like cells and human epithelial cells. The method is quick, efficient, and inexpensive, with no appreciable decrease in cellular viability. The introduced proteins can be visualized via immunofluorescence microscopy or used for functional assays. This has been demonstrated using green fluorescent protein (GFP) as a non-toxic standard, as well as two Salmonella effector proteins, SspH1 and GtgE. We propose protein electroporation as an additional tool in the repertoire for the study of bacterial virulence proteins and their functions in eukaryotic host cells.
1. Prepare in Advance
- Warm sterile phosphate buffered saline (PBS) to 37 °C.
- Warm Dulbecco’s Modification of Eagle’s Medium (DMEM) and Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 I.U./ml penicillin, and 100 µg/ml streptomycin to 37 °C. Note: These represent Normal Growth Media (NGM) for RAW and HeLa cells respectively.
2. Preparation of Cells
- Grow RAW 264.7 cells to 70-90% confluency in NGM.
- Maintain cells at 37 °C in a humidified 95% air/5% CO2 atmosphere.
- Grow HeLa cells to 70-90% confluency in NGM.
- Maintain cells at 37 °C in a humidified 95% air/5% CO2 atmosphere.
- Before collection, wash cell monolayer once with sterile PBS.
- Collect pre-confluent cells in a sterile conical tube.
- Gently scrape RAW cells with a rubber policeman in PBS, with repeated pipetting to disperse cell aggregations.
- Detach HeLa cells with 0.25% trypsin solution until visual examination shows dissociation from culture surface. For example, use 2 to 3 ml for a T-75 flask. Adjust volume accordingly to ensure trypsin solution covers entire growth surface.
- Quench dissociation reaction with NGM containing 10% FBS.
- Use at least twice the volume of NGM to trypsin, with repeated pipetting to disperse cell aggregations.
- Lightly pellet cells by centrifugation at 900 x g for 4 min.
- Resuspend in same volume as trypsin/quench solution using sterile PBS.
- Count cells using hemocytometer or particle counter.
- Lightly pellet 