rophage-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 cells by centrifugation at 900 x g for 4 min.
- Resuspend in adequate volume of PBS for 5.5 x 106 to 6.0 x 106 cells/ml. Note: For example, one T-75 flask will yield approximately 7.5 x 106 cells 29.
- Keep cell suspension on ice until electroporation.
3. Preparation for Electroporation
- Pre-chill electroporation cuvettes (0.4 cm gap) on ice.
- Turn on electroporation apparatus and set the voltage to 0.3 kV.
NOTE: Capacitance and resistance were not adjustable settings on our electroporator (set at 10 μF and 600 Ω by the manufacturer).
- Fill recovery plates with NGM and equilibrate in humidified 95% air/5% CO2 atmosphere at 37° C.
- Place 400 µl of cell suspension in pre-chilled cuvette and add 20 µg of selected protein to cuvette (50 µg/ml).
- Flick cuvette gently ~10 times to mix without damaging cells. Note: The cuvette may also be inverted several times to mix thoroughly, but do not pipet up and down or vortex to avoid damaging cells.
- Dry outside of cuvette with paper towel or other wiper to avoid electrical arcing in the electroporator.
- Electroporate sample at 0.3 kV for 1.5-1.7 msec. Note: This was typical for this study.
- Immediately after electroporation flick cuvette gently ~10 times to mix thoroughly.
5. Plating of Cells
- Store cuvette with electroporated cells on ice until ready to place in recovery plates.
- For most downstream analyses, wash cells 1x with pre-warmed NGM to remove extraneous effector protein.
- Remove cells for analysis and suspend in 3-5 ml of NGM.
- Pellet cells by centrifugation at 900 x g for 4 min.
- Resuspend in adequate volume of NGM for desired plate size (e.g. 2 ml for 35 mm dish).
- Remove appropriate amount of cells for downstream analysis.
- Example 1: plate into glass bottom dishes for microscopy analysis.
- Example 2: plate into cell culture plastic for protein analysis such as affinity purifications.
- Allow cells to recover in equilibrated plates in humidified 95% air/5% CO2 atmosphere at 37° C for at least 4 hr.
6. Microscopy Analysis
6.1) Fixation/Immunofluor