An Assay to Monitor Degranulation of Azurophilic Granules in Neutrophils following Activation
Transkript
Take tubes with mouse neutrophils suspended in a buffer.
These neutrophils contain intracellular granules, including primary, or azurophilic granules with myeloperoxidases.
Add N-formyl-methionyl-leucyl-phenylalanine or fMLP — a synthetic peptide, to one tube; the other serves as a control. Incubate.
fMLP binds to its specific receptor, triggering intracellular signaling, activating neutrophils, and increasing the cytoplasmic calcium concentration.
Further, the granules, including primary granules, translocate and fuse with the plasma membrane, mediated by calcium ions and membrane-bound proteins, resulting in increased degranulation into the buffer.
Post-incubation, centrifuge the tubes. Transfer the supernatant containing released cargo, including myeloperoxidase, into an assay plate.
Add hydrogen peroxide and tetramethylbenzidine. Incubate.
Myeloperoxidase utilizes hydrogen peroxide to oxidize tetramethylbenzidine, generating a blue-colored product.
Add an acidic solution to stop the reaction, changing the color to yellow.
Using a microplate reader, measure the product's absorbance, determining the concentration of secreted myeloperoxidase following neutrophil activation and degranulation.
