Name: Real-Time Polymorphonuclear Neutrophils (PMN) Chemotaxis Assay
Uploaded: 2024-02-09T12:40:00
Description: Watch this Scientific Journal Video about Real-Time Polymorphonuclear Neutrophils PMN Chemotaxis Assay at JoVE.com

real-time polymorphonuclear neutrophils (pmn) chemotaxis assay

Tests fonctionnels des neutrophiles pour le criblage de différentes voies de signalisation

Neutrophils are the most abundant innate immune cells in human blood and are known to play a major part in infection and inflammation, being the first responders to arrive at the site of tissue damage1. In recent years, there has been a growing recognition of the crucial role that neutrophils play in a variety of diseases and in supporting homeostasis2. Neutrophils not only have tightly regulated signaling themselves, but also participate in the regulation of other components of the immune system3,4,5. Therefore, investigating neutrophils and their many unusual cellular functions, such as chemotaxis, reverse migration6, phagocytosis7, respiratory burst8, and the release of neutrophil extracellular traps (NETs)7, is imperative in numerous research contexts where it is necessary to assess the potential neutrophil functional, morphological, or molecular changes triggered by specific conditions under analysis.
Freshly isolated neutrophils are terminally differentiated, short-lived, highly dynamic, and easily activated9. However, an efficient storage method that does not affect the neutrophil responses has not yet been achieved, making it challenging to perform multiple assays that must be uninterrupted. Furthermore, previously described functional analyses10,11, based on assays that require cytometry and/or fluorescent staining, may not be a viable choice when a broad and initial evaluation of the neutrophil is needed.
To address these issues, this protocol describes a set of tests that can be carried out following a single isolation process, including the evaluation of cell viability, reactive oxygen species (ROS) production, real-time migration, and phagocytosis of Saccharomyces cerevisiae, whose results can be used to reason in-depth follow-up studies. This procedure, named NeutroFun Screen, was designed to encompass the leading effector activities, except degranulation, and can be completed in an average time of 4 h, including 1 h of activation. Additionally, the remaining cells can be used for more detailed approaches like omics studies. The advantage of this method lies in its balance between speed, comprehensiveness, cost, and accuracy.
Furthermore, there is a way to easily observe a preliminary suggestion of NETs, without specific markers, but enough to indicate if further efforts in that direction would be worthwhile. The diversity of functions tested aims to combine common points among tests, reducing the analysis time and expenses. The main goal of this method is to provide a balanced, functional analysis regarding speed, comprehensiveness, cost, and accuracy that allows for an overview of the neutrophil&#39;s response, making it a useful initial step in investigating the effects of novel stimuli on normal density neutrophils.

Pleins feux sur l’auteur : Équilibrer la vitesse, le coût et la précision dans l’évaluation de la fonction des neutrophiles avec le protocole NeutroFun Screen 

Un ensemble de techniques de dépistage pour un aperçu rapide de la fonction des neutrophiles

The NeutroFun Screen Protocol provides a group of functional tests that use low-cost chemicals and can be performed in a single experiment from fresh neutrophils. This allows a preliminary evaluation of some major neutrophil functions to guide more laborious and expensive follow-up studies. Neutrophil research employs various modulation conditions.Such as cytokines, peptides, microbial products, and physical stimuli. Additionally, analyses flow cytometry, multiomics, microfluidic devices and immuno cytochemistry. The extensive brand of conditions and assays used in combination makes the workflow of neutrophil research long, laborious and expensive.Typically in these reduces, the number of conditions tested or the number of tests performed, the current state of the art methods require extensive configurables, many cells and lengthy preparation procedures. Our protocol NeutroFun Screen offers a quicker and more cost-effective approach to evaluate cell functionality compared to other methods. This is achieved by combining various analyses within the protocol, while maintaining accuracy and comprehensiveness in the results.Two researchers performed this workflow simultaneously for a quick overview of the neutrophil function. Researcher one isolates the PMNs, adjusts concentration and incubates the activation systems, whereas researcher two prepares materials for the subsequent cases. Then researcher one performs phagocytosis, and colorimetric NBT assays while researcher two conducts real-time migration and NBT slide tests.

Watch this Scientific Journal Video about Real-Time Polymorphonuclear Neutrophils PMN Chemotaxis Assay at JoVE.com

Real-Time Polymorphonuclear Neutrophils (PMN) Chemotaxis Assay  

Dosage de chimiotaxie des neutrophiles polymorphonucléaires (PMN) en temps réel

Préparez le gradient chimiotactique en ajoutant 160 microlitres de chimioattractants dans la chambre inférieure d’un analyseur de cellules en temps réel basé sur l’impédance, ou plaque RTCA. Pour les témoins négatifs et les blancs, ajoutez 160 microlitres de solution saline équilibrée ou HBSS de Hanks avec du calcium et du magnésium. Fixez la chambre supérieure et ajoutez 25 microlitres de HBSS avec du calcium et du magnésium.Incuber à température ambiante pendant au moins une heure pour former le gradient chimiotactique. Ensuite, mélangez délicatement et transférez 60 microlitres de polynutrophiles activés préalablement préparés dans la chambre supérieure. Ajoutez ensuite 60 microlitres d’HBSS avec du calcium et du magnésium à l’ébauche.Placez la plaque RTCA dans l’analyseur et programmez son logiciel pour mesurer l’indice de la cellule toutes les 60 secondes pendant deux heures. Ajustez la courbe avec les données obtenues. La migration cellulaire en temps réel a montré un effet chimiotactique de 100 nanomolaires fMLP sur les neutrophiles.

Research

JoVE Journal

Immunology and Infection

Real-Time Polymorphonuclear Neutrophils (PMN) Chemotaxis Assay

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as ...