Procedure
HeLa cells are commonly used as eukaryotic cells in the gentamicin protection assay, but other cells can be used as well. As for bacteria, only species susceptible to gentamicin can be assayed.
The assay is performed in polypropylene plates with round wells, which are commonly used in laboratories for culturing eukaryotic cells. The cells are allowed to grow in the wells overnight, creating a flat layer. Bacteria are separately grown overnight. On the next day the eukaryotic cells are inoculated with the bacteria and are incubated together for an hour. Centrifuging the plates for a few minutes may help bring cells and bacteria in contact and initiate infection.
After infection gentamicin is added to the plates, and they are incubated for an hour, allowing the antibiotic to kill all bacteria that were not able to penetrate the cells and remained outside. The plates are then washed well to remove the dead bacteria. Next the eukaryotic cells are lysed using a detergent, most commonly Triton X-100.
The bacteria that penetrated the cells and remained alive are now released, and they are plated on solid medium plates. Counting the colonies formed on the plates on the next day, and knowing how many bacteria were used in the beginning of the assay, enables the researcher to calculate the percentage of bacteria that were able to invade the eukaryotic cells.
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