A Degranulation Assay to Monitor Neutrophil Activation and Gelatinase Release
Transcript
Neutrophils are immune cells containing cytoplasmic granules that store proteases, including gelatinase.
Take tubes containing neutrophils in a growth medium.
Add N-formyl-methionyl-leucyl- phenylalanine or fMLP — a proinflammatory synthetic bacterial peptide — to one tube. The other tube serves as a control.
fMLP binds to its specific receptor on the neutrophil, triggering neutrophil activation.
The cytoplasmic granules migrate toward the plasma membrane. Membrane SNARE proteins mediate the granule and plasma membrane fusion, leading to cargo discharge and degranulation.
Centrifuge to pellet the cells. Transfer the gelatinase-containing supernatant to a multi-well plate containing a gelatinase substrate — a fluorophore-conjugated gelatin.
A high density of fluorophore labeling on the substrate causes intramolecular self-quenching of the fluorescence signal.
Gelatinase hydrolyzes the substrate, separating the fluorophore molecules and allowing fluorescence emission.
Using a microplate reader, measure fluorescence intensities in the wells to determine the concentration of released gelatinase following neutrophil activation and degranulation.
