A Degranulation Assay to Monitor Neutrophil Activation and Gelatinase Release

Abstract

This video demonstrates an assay to monitor neutrophil activation and granule exocytosis through a degranulation assay. Upon activation by proinflammatory compounds, cytoplasmic granules within neutrophils containing gelatinase fuse with the cell membrane, discharging their cargo. The presence of gelatinase in the supernatant is assessed through the hydrolysis of a fluorophore-conjugated gelatin substrate, confirming neutrophil activation and degranulation.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Prepare mouse bone marrow neutrophil Harvest bone marrow cells Bone marrow cells are flushed out from the femur and tibia of C57BL/6 mice with 10 ml Hank's balanced salt solution (HBSS) containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 0.1% bovine serum albumin (BSA), pH 7.4 using a 1…

Materials

EnzChek Gelatinase/Collagenase Assay kit	Thermo Fisher Scientific	E12055	Used for specific granule degranulation assay
Gibco™ RPMI1640	Thermo Fisher Scientific	21875034	Used for culturing neutrophils
BSA	Sigma	A3294	Used for culturing neutrophils
Percoll	Cytiva	17089101	Used for isolating neutrophils
HBSS	Thermo Fisher Scientific	14170112	Used for isolating and stimulating neutrophils
HEPES	Sigma	H4034	Used for isolating neutrophils
10x PBS	Thermo Fisher Scientific	70013032	Used for isolating neutrophils
Recombinant murine TNF-α	Peprotech	315-01A	Used for stimulating neutrophils
fMLP	Sigma	F3506	Used for stimulating neutrophils
PHERAstar FSX plate reader	BMG Labtech
Citrate-dextrose solution (ACD)	Sigma	C3821	Used for isolating human neutrophils
Dextran T500	Pharmacosmos	5510 0500 9007	Used for isolating human neutrophils