This study investigates the activation and degranulation of mouse neutrophils in response to fMLP, a synthetic peptide. The methodology includes measuring myeloperoxidase activity as an indicator of neutrophil activation.
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.
To begin, stimulate the mouse neutrophils in HBSS buffer containing 1 millimolar calcium chloride and magnesium chloride with or without fMLP or TNF-alpha for 10 minutes at 37 degrees Celsius.
Then, pellet the cells twice at 800 times g for 5 minutes. Collect 45 microliters of supernatant. Transfer the supernatant to the assay plate and run it in duplicates.
To detect the myeloperoxidase activity, add 80 microliters of 0.75 millimolar hydrogen peroxide solution, and 100 microliters of TMB solution to 20 microliters of supernatant. Then, incubate it at room temperature in the dark.
After 30 minutes, quench the reaction by adding 0.8 N hydrochloric acid. Read the absorbance on a PHERAstar plate reader at 450 nanometers. If necessary, generate a standard curve using recombinant myeloperoxidase to calculate the absolute amount of myeloperoxidase in the sample.
繁體中文
