The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Summary

Here, we present a protocol to detect the presence of neutrophils in fixed/permeabilized histology sections and assess the activation state of live purified neutrophils. In particular, the MUB₄₀ peptide binds lactoferrin present in neutrophil-specific and tertiary granules. Exposure of the granule contents through either permeabilization or neutrophil activation allows for the marking of neutrophils.

Abstract

Here, we provide a protocol involving the use of MUB₄₀, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high concentrations in specific and tertiary granules of neutrophils. This protocol details how MUB₄₀ conjugated directly to a fluorophore can be used to stain neutrophils in fixed/permeabilized tissues as well as how this can be used in live-cell imaging to assay for neutrophil activation and de-granulation. Neutrophil detection methods are limited to species-specific monoclonal antibodies, which are not always suitable for certain applications. MUB₄₀ does not penetrate the cell membrane and is thus excluded from lactoferrin stored in non-activated/non-permeabilized neutrophils. MUB₄₀ has the added benefit of recognizing lactoferrin from a broad host range, making it especially useful for comparing results in studies involving multiple research models, reducing the number of duplicate reagents, and simplifying protocols through single-step staining.

Introduction

Neutrophils are one of the primary arms of the innate immune system and are routinely recruited to the sites of inflammation around the body. The study of neutrophils has been largely impaired by their short lifespan in vitro (less than 8 h) and by limited detection tools under basal conditions or after activation. Here, we present a well-tested protocol for the broad and specific detection of mammalian neutrophils in fixed/permeabilized samples. We also provide a detailed protocol for staining live neutrophils with MUB₄₀. Using the live neutrophil staining protocol, the timing and location of neutrophil activation can be pinpointed. This protocol ....

Protocol

All methods described here have been reviewed and approved by the French organizations CoRC (Comité de Recherche Clinique), CPP (Comité de Protection des Personnes), and CNIL (Commission Nationale Informatique et Liberté).

1. Staining Neutrophils in Histopathology Tissue with Fluorescently Labelled MUB₄₀

1. To obtain the tissue for histopathology, fix tissue/biopsy sections in a suitable volume of 4% paraformaldehyde (PFA) for 30 min up to 4 h depending on th.......

Discussion

Here, two assays are described in which the MUB₄₀ peptide is used to study neutrophil inflammation and activation. We show how MUB₄₀ can reveal neutrophils present in histopathology sections or show their activation state using live purified neutrophils. The critical steps for using MUB₄₀ as a staining reagent are the same as with any other fluorescent detection method. Care must be taken to ensure compatibility of fluorescent signals and that adequate washing steps have been ta.......

Materials

Name	Company	Catalog Number	Comments
MUB40-Cy5	Benoit Marteyn	benoit.marteyn@pasteur.fr	Fluorescent MUB40 peptide (available with other conjugated fluorophores)
RI-MUB40-Cy5	Benoit Marteyn	benoit.marteyn@pasteur.fr	Retro-inverso fluorescent MUB40 peptide. Synthesized with D-amino acids and resistant to proteases (available with other conjugated fluorophores)
Parformaldehyde	Sigma-Aldrich	16005	Fixative for histology
Sucrose	Sigma-Aldrich	S7903	Solution used to remove excess PFA
Optimal Cutting Temperature Compound (OCT)	Sakura Finetek USA	4583	Used to freeze tissue before cryostat sectioning
2-Methylbutane	Sigma-Aldrich	M32631	Used to freeze tissue before cryostat sectioning
Ethanol	Sigma-Aldrich	1.00974	Used for dry ice bath to freeze tissue
Leica Cryostat	Leica Biosystems	CM1520	Used to section tissues
Glass microscopy slides	Fisher Scientific	12-518-101
Cover slips	Thor Labs	CG15CH	Hi precision coverslip
Dako pen	Sigma-Aldrich	Z377821	Used to prevent liquid loss during tissue staining
Saponin	Sigma-Aldrich	47036	Used to permeabilize cells for IF staining
DAPI	Sigma-Aldrich	10236276001	Stains DNA
Alexa fluor 488 Phalloidin	Fisher Scientific	A12379	Binds actin
Prolong Gold antifade Mountant	Fisher Scientific	P36930	Prolongs IF signal and resistance to photobleaching
Fluorescent microscope	Various	Various	Used to image IF slides
Anoxic Cabinet	Various	Various	Used for the purification of live inactivated neutrophils
Sodium Chloride NaCL	Sigma-Aldrich	S7653
EDTA	Sigma-Aldrich	E9884	Washing buffer component
MACS BSA buffer	Miltenyi Biotec	130-091-376	Washing buffer component
Percoll	Sigma-Aldrich	P4937	Gradient for neutrophil purification
CD235a glycophorin magnetic microbeads	Miltenyi Biotec	130-050-501	Used to remove contaminating RBCs
LS columns	Miltenyi Biotec	130-042-401	Used in the removal of RBCs from neutrophils
RPMI-1640 without Phenol Red	Sigma-Aldrich	R8755	Used for neutrophil assays