Name: Nitrotetrazolium Blue Chloride (NBT) Assay for Evaluating ROS Production
Uploaded: 2024-02-09T12:40:00
Description: Watch this Scientific Journal Video about Nitrotetrazolium Blue Chloride NBT Assay for Evaluating ROS Production at JoVE.com

nitrotetrazolium blue chloride (nbt) assay for evaluating ros production

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 Nitrotetrazolium Blue Chloride NBT Assay for Evaluating ROS Production at JoVE.com

Nitrotetrazolium Blue Chloride (NBT) Assay for Evaluating ROS Production  

Dosage du chlorure bleu de nitrotétrazolium (NBT) pour l’évaluation de la production de ROS

Commencez par dissoudre 0,0005 gramme de NBT dans 10 microlitres de DMSO. Après avoir vortex pendant 15 minutes, ajouter 90 microlitres de solution saline équilibrée de Hank ou HBSS avec du calcium et du magnésium et un microlitre des substances d’essai. Encore une fois, faites tourner le tube jusqu’à deux minutes.Pour effectuer l’essai sur lame NBT, mélangez et transférez délicatement deux microlitres des neutrophiles polymorphonucléaires activés préalablement préparés sur une lame de verre propre. Incuber la lame dans une chambre humidifiée à 37 degrés Celsius pendant 20 minutes. Ensuite, ajoutez un microlitre de la solution de travail NBT sur les cellules.Incuber à nouveau pendant 20 minutes à l’abri de la lumière. Après avoir séché la lame, fixez-la en appliquant une goutte de méthanol dans chaque puits pendant une minute. Colorer la lame avec 0,03 safranine pendant une minute supplémentaire.Dans chaque puits, comptez 100 cellules aléatoires, différenciant les neutrophiles avec et sans dépôts de formazan. Pour effectuer le test de spectrophotométrie NBT, mélangez délicatement et transférez 90 microlitres de neutrophiles polymorphonucléaires activés préalablement préparés dans un tube de microcentrifugation propre. Ensuite, ajoutez soigneusement 20 microlitres d’une solution NBT de six millimolaires et incubez le tube dans l’obscurité pendant 37 degrés Celsius pendant 20 minutes.Après incubation, ajouter 100 microlitres de 10%SDS et Vortex. Soniser avec une pointe de sonicate à 60% d’amplitude, en ajoutant cinq cycles de 15 secondes chacun et des intervalles de 15 secondes. Centrifuger à 12 000 g pendant cinq minutes.Transférez 60 microlitres de surnageant dans une plaque transparente à 96 puits et mesurez l’absorbance du produit surnageant à 570 nanomètres. Les résultats du test spectrophotométrique ont indiqué que 100 nanomolaires de PMA induisaient une production élevée de ROS par rapport au fMLP et aux groupes témoins. Les résultats des tests de lame NBT ont corroboré les résultats spectrophotométriques, montrant que le PMA était le stimulus induisant la production de ROS le plus intense par rapport au fMLP.

Research

JoVE Journal

Immunology and Infection

Nitrotetrazolium Blue Chloride (NBT) Assay for Evaluating ROS Production

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 ...

Recherche

Immunologie et infection

Neutrophil Isolation and Activation

Isolement et activation des neutrophiles

Neutrophil Phagocytosis Assay

Dosage de la phagocytose neutrophile

Real-Time Polymorphonuclear Neutrophils (PMN) Chemotaxis Assay

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

Neutrophil Extracellular Trap (NET) Suggestive Assay

Piège extracellulaire neutrophile (TNE) Dosage suggestif