An Assay to Monitor Degranulation of Azurophilic Granules in Neutrophils following Activation
Published: February 29, 2024
Abstract
This video demonstrates a degranulation assay of azurophilic granules in mouse neutrophils, involving the stimulation of neutrophils to release myeloperoxidase-containing azurophilic granules. The subsequent enzymatic reaction is employed to quantify myeloperoxidase levels, providing insight into neutrophil activation.
Protocol
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Prepare mouse bone marrow neutrophils 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 …
Divulgations
The authors have nothing to disclose.
Materials
| EnzChek Gelatinase/Collagenase Assay kit | Thermo Fisher Scientific | E12055 | Used for specific granule degranulation assay |
| 3,3',5,5'-tetramethylbenzidine Ready-to-Use solution | Thermo Fisher Scientific | J61325.EQE | Used for azurophilic granule degranulation assay |
| H2O2 | Millipore Sigma | H1009 | Used for azurophilic 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 |
Tags
Citer Cet Article
An Assay to Monitor Degranulation of Azurophilic Granules in Neutrophils following Activation. J. Vis. Exp. (Pending Publication), e22213, doi: (2024).