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

Farklı Sinyal Yollarının Tizlenmesi için Nötrofil Fonksiyonel Tahliller

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.

Yazar Spotlight: NeutroFun Ekran Protokolü ile Nötrofil Fonksiyon Değerlendirmesinde Hız, Maliyet ve Doğruluğun Dengelenmesi 

Nötrofil fonksiyonuna hızlı bir genel bakış için bir dizi tarama tekniği

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  

ROS Üretimini Değerlendirmek için Nitrotetrazolyum Mavi Klorür (NBT) Testi

0.0005 gram NBT'yi 10 mikrolitre DMSO'da çözerek başlayın. 15 dakika girdaplamadan sonra, 90 mikrolitre Hank'in Dengeli Tuz Çözeltisi veya kalsiyum ve magnezyum içeren HBSS ve bir mikrolitre test maddesi ekleyin. Yine, tüpü iki dakikaya kadar girdaplayın.NBT slayt testini gerçekleştirmek için, önceden hazırlanmış aktive edilmiş polimorfonükleer nötrofillerin iki mikrolitresini yavaşça karıştırın ve temiz bir cam slayt üzerine aktarın. Slaytı nemlendirilmiş odada 37 santigrat derecede 20 dakika inkübe edin. Daha sonra, hücrelerin üzerine bir mikrolitre NBT çalışma çözeltisi ekleyin.Işıktan koruyarak tekrar 20 dakika inkübe edin. Slaytı kuruttuktan sonra, her bir oyuğa bir dakika boyunca bir damla metanol uygulayarak sabitleyin. Slaytı bir dakika daha 0.03 safranin ile boyayın.Her kuyucukta, formazan birikintileri olan ve olmayan nötrofilleri ayırt eden 100 rastgele hücre sayın. NBT spektrofotometri testini gerçekleştirmek için, önceden hazırlanmış aktive edilmiş polimorfonükleer nötrofillerin 90 mikrolitresini yavaşça karıştırın ve temiz bir mikrosantrifüj tüpüne aktarın. Daha sonra dikkatlice 20 mikrolitre altı milimolar NBT çözeltisi ekleyin ve tüpü karanlıkta 37 santigrat derece boyunca 20 dakika inkübe edin.İnkübasyondan sonra 100 mikrolitre% 10 SDS ve Vortex ekleyin. Her biri 15 saniyelik beş döngü ve 15 saniyelik aralıklar ekleyerek% 60 genlikte bir uç sonikat ile sonikate. Beş dakika boyunca 12.000 g'da santrifüjleyin.60 mikrolitre süpernatanı 96 oyuklu şeffaf bir plakaya aktarın ve süpernatan ürünün emilimini 570 nanometrede ölçün. Spektrofotometrik testin sonuçları, 100 nanomolar PMA'nın fMLP ve kontrol gruplarına kıyasla yüksek ROS üretimini indüklediğini gösterdi. NBT slayt testi sonuçları, PMA'yı fMLP'ye kıyasla en yoğun ROS üretimini indükleyen uyaran olarak gösteren spektrofotometrik sonuçları doğruladı.

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 ...