Visualization of Caspase Activation in Human Monocyte-Derived Macrophages
내레이션 대본
In immune cells, caspase-1, an inflammatory caspase, is a key immune-responsive component that exists in an inactive form. These inactive forms consist of a pro-domain fused with a catalytic domain and become active via proximity-induced dimerization.
To visualize caspase activation in vivo, begin with a culture of transfected human monocyte-derived macrophages expressing red fluorescent proteins and a pair of fluorescent complementation reporter proteins. The reporter proteins contain a pro-domain of caspase-1 fused with non-fluorescent fragments of the Venus protein, either Venus-N or Venus-C.
Treat the cells with an imaging medium containing lipopolysaccharides — a bacterial-derived inflammatory stimulant, and potassium ionophores — nigericin.
Lipopolysaccharides interact with pattern recognition receptors on the macrophage, initiating the assembly of an inflammasome complex.
Nigericin molecules enter the cell and bind to potassium ions, causing their efflux into the extracellular medium and decreasing the intracellular potassium ion concentration. This leads to the priming of inflammasome sensor proteins with adaptor proteins — a bridging protein between sensor protein and procaspase-1, forming the complex.
The pro-domains containing Venus fragments interact with the complex's adaptor proteins. This brings the two fragments in proximity, forcing them to refold and fluoresce.
Visualize the cells under an epifluorescence microscope.
The transfected cells appear red with green fluorescence spots, confirming caspase activation.
