Isolation of Intracellular Pathogenic Bacteria from Infected Host Cells

Take a suspension of pathogenic bacteria.

Add the suspension to an adhered culture of bone marrow-derived macrophage or BMDM cells and incubate.

The bacteria use their surface proteins to bind to host cell receptors, facilitating adherence.

Rinse with a buffer to remove unadhered bacteria.

Add fresh media and incubate.

The adhered bacteria are then internalized by the host cell.

Add an antibiotic to kill non-internalized bacteria.

Discard the media and wash with buffer.

Add chilled water to lyse the host cell, releasing cellular contents, the nucleus, and internalized bacteria.

Collect the cell lysate and centrifuge to precipitate cellular debris.

Collect the supernatant containing the bacteria. Pass it through a filtration device to capture intact bacteria.

Fold the filter paper and place it into a tube.

Flash-freeze in liquid nitrogen to preserve cellular integrity.

Store under cold conditions for further use.

Using 0.5 milliliters of the washed bacterial suspension, infect each macrophage plate. If using multiple plates, space the infections 15 minutes apart to allow for harvesting each plate individually at the end of the infection. Following a 30-minute incubation at 37 degrees Celsius, use