Introduction of Bacterial Effector Proteins into Mammalian Cells by Electroporation

Abstract

Source: Sontag, R. L. et al., Electroporation of Functional Bacterial Effectors into Mammalian Cells. J. Vis. Exp. (2015)

In this video, we demonstrate the introduction of bacterial effector proteins into mammalian cells by electroporation. The intracellular localization of the bacterial effector proteins is detected by confocal microscopy following staining with specific fluorophore-conjugated antibodies.

Protocol

1. Electroporation

1. Place 400 µl of cell suspension in a pre-chilled cuvette and add 20 µg of selected protein to the cuvette (50 µg/ml).
2. Flick the 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.
3. Dry the outside of the cuvette with a paper towel or other wiper to avoid electrical arcing in the electroporator.
4. Electroporate sample at 0.3 kV for 1.5-1.7 msec. Note: This was typical for this study.
5. Immediately afte....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA Solution	Cellgro	25-050-Cl
0.4cm Gap-disposable electroporation cuvettes	Bio-Rad	165-2088
100% Methanol	Any	N/A	Flammable, Toxic
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A4919
Cell Counting Apparatus - Hemocytometer or Coulter Counter	Beckman Coulter	Model Z1
Cell Culture Incubator	Any	N/A	Humidified 95% air/5% CO2 atmosphere at 37 °C
Cell Culture Plastic	Any	N/A	Cell culture flasks/plates, pipets, tubes, rubber policeman
Dulbecco's Modification of Eagle’s Medium (DMEM)	Cellgro	10-013	Warm to 37 °C
Electroporator	Bio-Rad	E. coli Pulser
Fetal Bovine Serum (FBS)	Cellgro	35-016-CV
Fluorescent confocal microscope	Ziess	Model LSM 710
Glass Bottom Dishes for Microscopy	Wilco Wells	HBSt-3522
HeLa Cell Line	ATCC	ATCC CCL-2
Minimal Essential Medium (MEM)	Cellgro	10-010	Warm to 37 °C
Other fluorescent stains (WGA, DAPI) in conjunction with anti-fade reagent	Any	N/A
Penicillin/Streptomycin	Cellgro	30-002-Cl
RAW 264.7 Cell line	ATCC	TIB-71
Primary Antibody Against Target of Interest	Any	N/A
Secondary Antibody Conjugated to Fluorophore	Any	N/A
Phosphate-Buffered Saline	Any	N/A	Chill to 4 °C
Sterile Phosphate-Buffered Saline	Any	N/A	Warm to 37 °C
Table Top Centrifuge Capable of Accepting Conical Tubes (swinging bucket preferred)	Any	N/A
Triton X-100	Sigma-Aldrich	T8585	Irritant, Toxic