An Assay to Monitor Degranulation of Azurophilic Granules in Neutrophils following Activation
Trascrizione
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.
