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

다양한 신호전달 경로를 스크리닝하기 위한 호중구 기능 분석

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.

저자 스포트라이트: NeutroFun Screen Protocol을 사용한 호중구 기능 평가의 속도, 비용 및 정확성 균형 맞추기 

호중구 기능에 대한 간략한 개요를 위한 일련의 스크리닝 기법

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 생산 평가를 위한 니트로테트라졸륨 블루 클로라이드(NBT) 분석

0.0005 마이크로리터의 DMSO에 NBT 10g을 용해시키는 것으로 시작합니다. 15분 동안 소용돌이친 후 칼슘과 마그네슘이 함유된 Hank's Balanced Salt Solution 또는 HBSS 90마이크로리터와 테스트 물질 1마이크로리터를 추가합니다. 다시 말하지만, 최대 2분 동안 튜브를 소용돌이치게 합니다.NBT 슬라이드 테스트를 수행하려면 이전에 준비한 활성화된 다형핵 호중구 2마이크로리터를 부드럽게 혼합하여 깨끗한 유리 슬라이드에 옮깁니다. 섭씨 37도의 가습 챔버에서 20분 동안 슬라이드를 배양합니다. 다음으로, 셀 위에 NBT 작업 용액 1마이크로리터를 추가합니다.빛으로부터 보호하면서 20분 동안 다시 배양합니다. 슬라이드를 건조시킨 후 각 웰에 메탄올 한 방울을 1분 동안 도포하여 고정합니다. 슬라이드에 0.03 사프라닌을 추가로 1분 동안 묻힙니다.각 웰에서 100개의 무작위 세포를 세어 포르마잔 침전물이 있는 호중구와 없는 호중구를 구별합니다. NBT 분광광도법 분석을 수행하려면 이전에 준비된 활성화된 다형핵 호중구 90마이크로리터를 부드럽게 혼합하여 깨끗한 미세원심분리기 튜브로 옮깁니다. 그런 다음 20 마이크로 리터의 6 밀리몰 NBT 용액을 조심스럽게 넣고 어두운 곳에서 섭씨 37도에서 20 분 동안 튜브를 배양합니다.배양 후 100 마이크로 리터의 10 % SDS와 Vortex를 첨가하십시오. 60 % 진폭에서 팁 초음파 처리로 초음파 처리하여 각각 15 초와 15 초 간격의 5 사이클을 추가합니다. 12에 분리기, 5 분 동안 000g.60마이크로리터의 상층액을 투명한 바닥 96웰 플레이트로 옮기고 570나노미터에서 상층액 생성물의 흡광도를 측정합니다. 분광광도계 분석 결과 100 나노몰 PMA가 fMLP 및 대조군에 비해 ROS 생산량 증가를 유도한 것으로 나타났습니다. NBT 슬라이드 테스트 결과는 분광광도계 결과를 확증하여 PMA가 fMLP에 비해 가장 강력한 ROS 생성 유도 자극임을 보여주었습니다.

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 ...