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 …

Disclosures

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

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Cite This Article
An Assay to Monitor Degranulation of Azurophilic Granules in Neutrophils following Activation. J. Vis. Exp. (Pending Publication), e22213, doi: (2024).

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