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.
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Prepare mouse bone marrow neutrophil
2. Isolation of human neutrophils
3. Specific granule degranulation assay
Table 1. Component of Percoll gradient solution.
55% Percoll (30 ml) | 74% Percoll (30 ml) | |
Percoll (ml) | 16.5 | 22.2 |
9% NaCl (ml) | 1.83 | 2.47 |
0.9% NaCl (ml) | 11.67 | 5.33 |
The authors have nothing to disclose.
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 |