A Degranulation Assay to Monitor Neutrophil Activation and Gelatinase Release

Published: February 29, 2024

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…

Açıklamalar

The authors have nothing to disclose.

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

Etiketler

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A Degranulation Assay to Monitor Neutrophil Activation and Gelatinase Release. J. Vis. Exp. (Pending Publication), e22212, doi: (2024).

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