A Degranulation Assay to Monitor Neutrophil Activation and Gelatinase Release

To begin, stimulate the neutrophils in RPMI1640, with or without 0.5 or 10 micromolar fMLP or 5 to 20 nanograms per milliliter TNF-alpha, and incubate for 10 minutes at 37 degrees Celsius.

At the end of the incubation, centrifuge the cells at 800 times g for 5 minutes. Collect the supernatant, and spin again, as demonstrated earlier.

To measure the specific granule degranulation, use the EnzChek gelatinase-collagenase assay kit. As per the manufacturer's instructions, add 1 milliliter of Milli-Q water to the lyophilized DQ gelatin vial to make 1 milligram per milliliter of DQ gelatin stock solution.

Then, prepare the reaction buffer by mixing 18 milliliters of Milli-Q water and 2 milliliters of 10X reaction buffer into a 15-milliliter tube. Add 0.5 milliliters of Milli-Q water to the Clostridium collagenase tube to prepare a 1,000 units per milliliter stock solution.

Before the experiment, dilute the collagenase standard solution using the reaction buffer to make different concentrations for generating the standard curve.

Next, add 80 microliters of the reaction buffer to each well of a 96-well plate. Dilute DQ gelatin stock solution four times using the reaction buffer before use. Then, add 20 microliters of diluted DQ gelatin solution to each well.

Add 100 microliters of sample or different concentrations of Clostridium collagenase standard solution into each well.

For data collection, insert the assay plate into the PHERAstar plate reader. Then, represent the data as a total activity using known concentrations of collagenase as a standard curve.