Introduction of Bacterial Effector Proteins into Mammalian Cells by Electroporation

Certain gram-negative bacterial pathogens contact host cells and employ secretion systems to inject effector proteins, facilitating cell invasion.

To deliver effector proteins into mammalian cells in vitro, pipette mammalian cells into a chilled cuvette. Add streptavidin-binding peptide-tagged bacterial effector proteins and mix.

Perform electroporation. During the run, short electric pulses disrupt the host cell membrane lipid bilayer to form transient pores, facilitating the cellular entry of effector proteins. Plate the cells onto a glass plate containing media, allowing the cells to recover and adhere to the plate. The pores reseal, entrapping the proteins within.

Following cell recovery, fix and permeabilize the cells for antibody access to intracellular targets. Incubate with a protein-containing blocking solution to prevent non-specific antibody binding. Add primary antibodies that specifically bind to the streptavidin-binding peptide tag on the intracellular effector proteins.

Introduce fluorophore-tagged secondary antibodies that bind to primary antibodies bound to effector proteins for visualization. Add fluorophore-conjugated wheat germ agglutinin and DAPI to stain cell membrane glycoproteins and DNA, respectively.

Using a confocal microscope, visualize fluorescence signals from effector proteins, cell membranes, and DNA.

Successfully electroporated cells exhibit significant intracellular fluorescent signals from effector proteins, suggesting their intracellular localization.

To begin this procedure, transfer 400 microliters of cell suspension to a pre-chilled cuvette, then, add 20 micrograms of selected protein to reach a final concentration of 50 micrograms per milliliter. Flick the cuvette gently, about 10 times for mixing and to avoid damaging the cells. After that, dry the outside of the cuvette with a paper towel to avoid electrical arcing in the electroporator.

Place the sample in the electroporator and set it to 0.3 kilovolts for 1.5 to 1.7 milliseconds. Immediately after electroporation, flick the cuvette gently, about 10 times, to mix the sample thoroughly before proceeding to either fixation and immunofluorescence staining or affinity purification.

After four hours of recovery from electroporation, wash the cells with sterile PBS. Then, fix the cells in 100% methanol for two minutes at room temperature. Subsequently, wash the cells with sterile PBS three more times. Permeabilize the cells with 0.4% Triton X-100 in PBS for 15 minutes. Adjust the length of permeabilization and the strength of Triton X-100 according to the epitope and location of the target protein. Then, block the cells with 5% BSA in PBS for one hour at room temperature.

Afterward, wash them three times with PBS. Next, incubate them with the primary antibody in the antibody-binding solution overnight, at 4 degrees Celsius with gentle rocking.

The next day, wash the cells four more times with PBS. Then, incubate them with the appropriate fluorescently-conjugated secondary antibody in the antibody-binding solution. Add other stains as needed, such as 5 micromolar Wheat Germ Agglutinin, or WGA, conjugated to Alexa 647 or DAPI for one hour at room temperature and protected from light. Then, wash the cells with PBS five times and store at 4 degrees Celsius, protected from light until they are ready to be imaged.