This article details the activation and degranulation of neutrophils, highlighting the role of gelatinase in this process. The methodology involves stimulating neutrophils with fMLP and measuring gelatinase activity through fluorescence.
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.
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.
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