This article details a method for studying the interaction between pathogenic bacteria and bone marrow-derived macrophages (BMDMs). The protocol outlines steps for infection, internalization, and subsequent analysis of bacteria within 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 PBS to wash the infected cells twice to remove unattached bacteria, and then add 30 milliliters of pre-warmed BMDM medium.
Incubate for 30 minutes to allow bacteria to internalize. At one hour post infection, add gentamicin to a final concentration of 50 micrograms per milliliter to kill any remaining extracellular bacteria. Assemble the filter apparatus by placing a filter head on a collecting liquid flask with a vacuum outlet port.
Then place a 0.45 micron filter on the filter head, followed by a cylinder funnel, and use a metal clamp to secure the different parts. At six hours post infection, examine the cell monolayer under the microscope before harvesting. Working with one plate at a time to harvest the bacteria, use PBS to wash the infected cells.
Then, to lyse the macrophage cells, add 20 milliliters of ice-cold RNAse-free water, and quickly but carefully use a cell scraper to scrape the cells off the plate. After collecting the cells into a 50 milliliter conical tube, vortex for 30 seconds, then centrifuge at 800 times g and 4 degrees Celsius for 3 minutes to remove macrophage cell nuclei. To collect bacteria, pass the supernatant through the filter using the vacuum system.
Then, with tweezers, roll the filter and quickly transfer it to a 15 milliliter conical tube. Immediately use liquid nitrogen to snap freeze the tube.
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