Encyclopedia of Experiments: Immunology
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Transcript
Take wells containing polyacrylamide hydrogels of varying stiffness overlaid with collagen, mimicking a physiological extracellular matrix, or ECM.
Add microvascular endothelial cells that form focal adhesions, or FAs — complexes mediating cell-ECM binding — to sense the hydrogel stiffness.
Stiffer hydrogels elevate cell-surface expression of vimentin, a cytoskeletal component, compared to softer hydrogels.
Add Listeria monocytogenes bacteria, engineered to express a fluorescent protein under the promoter for actin assembly-inducing protein, or ActA.
Centrifuge to synchronize bacterial contact with the host cells, and incubate.
The bacteria bind to vimentin. More vimentin on the stiffer hydrogel-bound host cells leads to more bacterial adherence and endocytosis.
Add an antibiotic, eliminating non-internalized bacteria.
The internalized bacteria secrete toxins, rupturing the endosome membrane. Cytoplasmic escape activates the ActA promoter to express the fluorescent protein, labeling intracellular bacteria.
Enzymatically detach the cells. Using flow cytometry, detect a higher fluorescence in host cells residing on stiffer hydrogels, indicating greater bacterial internalization.
