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.
1. Electroporation
2. Plating of Cells
3. Microscopy Analysis
The authors have nothing to disclose.
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 |