Encyclopedia of Experiments: Immunology
A subscription to JoVE is required to view this content.
Transcript
Take a multi-well plate containing a macrophage culture. Infect with fluorescent protein-expressing Listeria monocytogenes, Lm.
Proteins on Lm bind to specific macrophage receptors, internalizing the bacteria within a phagosome. Additionally, Lm secretes pore-forming toxins that disrupt the phagosome membrane, releasing bacteria into the cytosol, where they replicate and become surrounded by host actin filaments.
The actin assembly-inducing, ActA proteins on Lm induce continuous actin polymerization at the bacterium's rear end. The generated actin comet tail propels Lm through the cytosol and penetrates the macrophage cell membrane, releasing polymerized actin-enveloped Lm into the media.
Post-incubation, collect and fix the extracellular, actin-bound, Lm-containing media from a set of wells. Fix the Lm using paraformaldehyde. Add distilled water, rupturing the macrophages and releasing the intracellular Lm. Collect the intracellular Lm and fix the sample using paraformaldehyde.
In other wells, replace media with cell-impermeable gentamicin-containing media to eliminate extracellular Lm. Replace with fresh media and incubate for the desired period.
Post-incubation, collect the extracellular Lm-containing media and fix the sample with paraformaldehyde. Use distilled water to obtain lysate containing intracellular Lm and fix the sample with paraformaldehyde. Add fluorescently labeled phalloidin to the samples, which selectively binds to polymerized actin.
Using a flow cytometer, visualize fluorescent signals from Lm and actin-binding phalloidin in the samples. Quantify the extracellular and intracellular actin-bound Lm derived from the infected macrophages.
