eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Strains and culture conditions
Note: M. tuberculosis strain CDC1551 is used in this study, but any M. tuberculosis strain can be used in the same manner.
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	<li>Grow the bacteria in M-OADC-TW (7H9 broth supplemented with 0.5% glycerol, 10% oleic acid dextrose compl...

reporter enzyme fluorescence imaging for in vivo tracking of bacterial infection

Source: Sharan, R., et al. <a href="https://app.jove.com/v/56801">Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence.</a> J. Vis. Exp. (2018).
This video demonstrates the reporter enzyme fluorescence (REF) imaging for in vivo detection of Mycobacterium tuberculosis infection. The infected mice are intraperitoneally injected with a substrate for REF imaging. Upon being hydrolyzed by bacterial &#946;-lactamase enzyme, the substrate emits fluorescence, the detection of which aids evaluation of infection.

Reporter Enzyme Fluorescence Imaging for In Vivo Tracking of Bacterial Infection

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Expose a mouse to aerosol droplets containing Mycobacterium tuberculosis.
Bacteria reach the lumen of alveoli &#8212; the tiny air sacs within the lungs &#8212; containing macrophages.
Macrophage pattern recognition receptors bind to the pathogen-associated molecular patterns on the bacteria, triggering their engulfment into a phagosome.
Engulfed bacteria secrete specialized proteins, which rupture the phagosome membrane. The bacteria escape into the cytoplasm and reproduce.
Post-infection, anesthetize the mouse. Intraperitoneally administer a reporter substrate of the bacterial &#946;-lactamase enzyme for reporter enzyme fluorescence or REF imaging.
The substrate reaches lung alveoli via circulation and enters the infected cells.
It penetrates the bacterial outer membrane to reach the periplasmic space containing &#946;-lactamase.
The substrate contains a &#946;-lactam ring connecting a fluorochrome and a quencher. Due to proximity, the fluorochrome&#39;s fluorescence is absorbed by the quencher.
&#946;-lactamase hydrolyzes the &#946;-lactam ring, releasing the fluorophore and resulting in fluorescence emission.
Image the fluorescence to assess mycobacterial infection.