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

さまざまなシグナル伝達経路をスクリーニングするための好中球機能アッセイ

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.

著者スポットライト:NeutroFunスクリーンプロトコルによる好中球機能評価における速度、コスト、精度のバランス 

好中球機能の概要を把握するための一連のスクリーニング技術

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産生評価のためのニトロテトラゾリウム青色塩化物(NBT)アッセイ

まず、0.0005グラムのNBTを10マイクロリットルのDMSOに溶解します。15分間ボルテックスした後、カルシウムとマグネシウムを含む90マイクロリットルのハンク平衡塩溶液またはHBSSと1マイクロリットルの試験物質を加えます。ここでも、チューブを最大2分間ボルテックスします。NBTスライド試験を実施するには、事前に調製した活性化多形核好中球2マイクロリットルを静かに混合し、清潔なスライドガラスに移します。スライドを摂氏37度の加湿チャンバーで20分間インキュベートします。次に、1マイクロリットルのNBT作業溶液を細胞上に加えます。再び20分間インキュベートし、光から保護します。スライドを乾燥させた後、各ウェルにメタノールを1滴垂らして固定します。スライドを0.03サフラニンでさらに1分間染色します。各ウェルで、100個のランダムな細胞を数え、ホルマザン沈着物の有無にかかわらず好中球を区別します。NBT分光光度法アッセイを実施するには、事前に調製した90マイクロリットルの活性化多形核好中球を静かに混合し、清潔な微量遠心チューブに移します。次に、20マイクロリットルの6ミリモルのNBT溶液を慎重に加え、チューブを暗所で摂氏37度で20分間インキュベートします。インキュベーション後、100マイクロリットルの10%SDSとVortexを加えます。チップで超音波処理を行い、60%の振幅で超音波処理を行い、それぞれ15秒間隔のサイクルを5回追加します。12 、 000 g で 5 分間遠心分離します。60マイクロリットルの上清を透明な底の96ウェルプレートに移し、570ナノメートルで上清生成物の吸光度を測定します。分光光度法の結果は、100ナノモルのPMAがfMLPおよび対照群と比較してROS産生の上昇を誘発することを示しました。NBTスライドテストの結果は、分光光度法の結果を裏付け、PMAがfMLPと比較して最も強いROS産生誘導刺激であることを示しました。

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 ...