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

Neutrofile funktionelle analyser til screening af forskellige signalveje

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.

Forfatter Spotlight: Balancering af hastighed, omkostninger og nøjagtighed i vurdering af neutrofil funktion med NeutroFun Screen Protocol 

Et sæt screeningsteknikker til et hurtigt overblik over neutrofilfunktionen

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  

Nitrotetrazoliumblåchlorid (NBT) assay til evaluering af ROS-produktion

Begynd med at opløse 0,0005 gram NBT i 10 mikroliter DMSO. Efter hvirvelstrøm i 15 minutter tilsættes 90 mikroliter Hank's Balanced Salt Solution eller HBSS med calcium og magnesium og 1 mikroliter af teststofferne. Igen, hvirvel røret i op til to minutter.For at udføre NBT-diastesten blandes forsigtigt og overføres to mikroliter af de tidligere fremstillede aktiverede polymorfonukleære neutrofiler på et rent glasglas. Inkuber objektglasset i befugtet kammer ved 37 grader Celsius i 20 minutter. Derefter tilsættes en mikroliter af NBT-arbejdsløsningen over cellerne.Inkuber igen i 20 minutter og beskyt mod lys. Efter tørring af diaset skal du rette det ved at påføre en dråbe methanol i hver brønd i et minut. Plet diaset med 0,03 safranin i yderligere et minut.I hver brønd tæller du 100 tilfældige celler, der differentierer neutrofiler med og uden formazanaflejringer. For at udføre NBT-spektrofotometrianalysen blandes og overføres forsigtigt 90 mikroliter af de tidligere fremstillede aktiverede polymorfonukleære neutrofiler til et rent mikrocentrifugerør. Derefter tilsættes forsigtigt 20 mikroliter af en seks millimolær NBT-opløsning og inkuberer røret i mørke i 37 grader Celsius i 20 minutter.Efter inkubation tilsættes 100 mikroliter 10% SDS og hvirvel. Sonikere med en spids sonikere ved 60% amplitude, tilføje fem cyklusser på 15 sekunder hver og 15 sekunders intervaller. Centrifuger ved 12.000 g i fem minutter.60 mikroliter supernatant overføres til en plade med en klar bundplade med 96 huller, og supernatantproduktets absorbans måles til 570 nanometer. Resultaterne af det spektrofotometriske assay indikerede, at 100 nanomolær PMA inducerede forhøjet ROS-produktion sammenlignet med fMLP og kontrolgrupperne. NBT-diastestresultaterne bekræftede de spektrofotometriske resultater og viste PMA som den mest intense ROS-produktionsinducerende stimulus sammenlignet med 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 ...