JoVE Encyclopedia of Experiments
Immunology
0 views • 5:13 min • July 8th, 2025
Take a multi-well plate containing adhered epithelial cells and treat it with green fluorescent protein-tagged Coxiella mutants; some with mutations in the type IV secretion system gene, or T4SS gene — crucial for replication.
Replicative mutants interact with epithelial cells, triggering Coxiella engulfment, forming Coxiella-containing vacuole, or CCV, and recruiting lysosome-associated membrane glycoprotein-1, or LAMP1 to CCV.
This facilitates effector proteins' secretion into the cell cytoplasm via the type IV secretion system, promoting bacterial replication.
Mutants with T4SS gene mutations remain unreplicated.
Post-incubation, stain the cells with a fluorescent dye, fix them, and permeabilize the cell membrane.
Introduce LAMP1-specific primary antibodies interacting with the LAMP1 proteins on CCV.
Overlay with red-fluorophore-labeled secondary antibodies, interacting with primary antibodies, while Hoechst dye stains the cells' nuclei.
Under an epifluorescence microscope, red-fluorescence vacuoles containing fewer green Coxiella near the nucleus indicate defective replication of Coxiella mutants.
However, vacuoles containing many green Coxiella inside the cells indicate the s
View the full transcript and gain access to thousands of scientific videos