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Cell Biology JoVE (Open Access) Citable · DOI

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

DOI: 10.3791/50116-v
What you'll learn
  • ✓ Perform CCF4-AM/β-lactamase assay to detect vacuolar rupture in fixed cells
  • ✓ Apply live-cell CCF4 imaging to track intracellular pathogen invasion dynamics
  • ✓ Interpret FRET signal loss as marker of bacterial endomembrane degradation
Protocol

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Difficulty
intermediate
Total time
~4-6 hours per sample batch (includes cell infection, staining, imaging, and image analysis)
Model organism
HEK293 (human epithelial cells); Shigella flexneri and Mycobacterium tuberculosis (intracellular bacteria)
Biosafety
BSL-2

Steps

1
Perform CCF4 assay on fixed Shigella-infected cells

Load CCF4-AM into fixed HEK293 cells infected with Shigella flexneri in 96-well plates. Bacterial β-lactamase degrades the FRET probe in the cytoplasm, reducing blue fluorescence and increasing green signal to indicate vacuolar rupture.

▶ 01:29
2
Conduct live-cell CCF4 imaging of Shigella invasion

Apply CCF4-AM to live HEK293 cells infected with Shigella in 35 mm MatTek glass-bottom dishes. Monitor real-time FRET signal loss via fluorescence microscopy to track temporal dynamics of vacuolar membrane rupture during bacterial infection.

▶ 05:31
3
Interpret CCF4 FRET data and pathogen detection results

Analyze quantitative fluorescence readouts comparing infected versus uninfected controls. FRET loss indicates successful bacterial escape into cytoplasm and confirms β-lactamase activity from pathogen surface.

▶ 07:04
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