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…
Disclosures
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 |
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Cite This Article
A Degranulation Assay to Monitor Neutrophil Activation and Gelatinase Release. J. Vis. Exp. (Pending Publication), e22212, doi: (2024).