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

Ensaios funcionais de neutrófilos para triagem de diferentes vias de sinalização

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.

Autor em destaque: equilibrando velocidade, custo e precisão na avaliação da função de neutrófilos com o protocolo de tela NeutroFun 

Um conjunto de técnicas de triagem para uma rápida visão geral da função dos neutrófilos

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  

Ensaio de cloreto de azul de nitrotetrazólio (NBT) para avaliação da produção de ROS

Comece dissolvendo 0,0005 gramas de NBT em 10 microlitros de DMSO. Após vórtice por 15 minutos, adicionar 90 microlitros de Hank's Balanced Salt Solution ou HBSS com cálcio e magnésio e um microlitro das substâncias de teste. Novamente, agite o tubo por até dois minutos.Para realizar o teste de lâmina NBT, misture e transfira suavemente dois microlitros dos neutrófilos polimorfonucleares ativados previamente preparados em uma lâmina de vidro limpa. Incubar a lâmina em câmara umidificada a 37 graus Celsius por 20 minutos. Em seguida, adicione um microlitro da solução de trabalho NBT sobre as células.Incubar novamente por 20 minutos, protegendo da luz. Depois de secar a lâmina, fixe-a aplicando uma gota de metanol em cada poço por um minuto. Manchar a lâmina com 0,03 safranina por mais um minuto.Em cada poço, contar 100 células aleatórias, diferenciando neutrófilos com e sem depósitos de formazana. Para realizar o ensaio de espectrofotometria NBT, misture e transfira suavemente 90 microlitros dos neutrófilos polimorfonucleares ativados previamente preparados para um tubo de microcentrífuga limpo. Em seguida, adicione cuidadosamente 20 microlitros de uma solução de NBT de seis milimolares e incube o tubo no escuro por 37 graus Celsius por 20 minutos.Após a incubação, adicione 100 microlitros de 10% SDS e Vortex. Sonicate com uma ponta sonicate a 60% de amplitude, adicionando cinco ciclos de 15 segundos cada e intervalos de 15 segundos. Centrifugar a 12.000g por cinco minutos.Transfira 60 microlitros de sobrenadante para uma placa de fundo transparente de 96 poços e meça a absorbância do produto sobrenadante a 570 nanômetros. Os resultados do ensaio espectrofotométrico indicaram que 100 PMA nanomolares induziram elevada produção de ROS em comparação com os grupos fMLP e controle. Os resultados do teste de lâminas NBT corroboraram os resultados espectrofotométricos, mostrando PMA como o estímulo indutor de produção de ERO mais intenso em comparação com 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 ...