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

Ensayos funcionales de neutrófilos para el cribado de diferentes vías de señalización

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 destacado: Equilibrio de la velocidad, el costo y la precisión en la evaluación de la función de los neutrófilos con el protocolo de detección NeutroFun 

Un conjunto de técnicas de cribado para una visión general rápida de la función de los 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  

Ensayo de cloruro azul de nitrotetrazolio (NBT) para evaluar la producción de ROS

Comience disolviendo 0,0005 gramos de NBT en 10 microlitros de DMSO. Después de agitar el vórtice durante 15 minutos, agregue 90 microlitros de la solución salina equilibrada de Hank o HBSS con calcio y magnesio y un microlitro de las sustancias de prueba. De nuevo, haz vórtice en el tubo durante un máximo de dos minutos.Para realizar la prueba de portaobjetos NBT, mezcle suavemente y transfiera dos microlitros de los neutrófilos polimorfonucleares activados previamente preparados a un portaobjetos de vidrio limpio. Incubar el portaobjetos en una cámara humidificada a 37 grados centígrados durante 20 minutos. A continuación, agregue un microlitro de la solución de trabajo NBT sobre las células.Incubar de nuevo durante 20 minutos, protegiendo de la luz. Después de secar el portaobjetos, fíjelo aplicando una gota de metanol en cada pocillo durante un minuto. Tiñe el portaobjetos con 0,03 safranina durante un minuto más.En cada pocillo, cuente 100 células al azar, diferenciando los neutrófilos con y sin depósitos de formazano. Para realizar el ensayo de espectrofotometría NBT, mezcle suavemente y transfiera 90 microlitros de los neutrófilos polimorfonucleares activados previamente preparados a un tubo de microcentrífuga limpio. A continuación, agregue con cuidado 20 microlitros de una solución NBT de seis milimolares e incube el tubo en la oscuridad durante 37 grados centígrados durante 20 minutos.Después de la incubación, agregue 100 microlitros de SDS al 10% y Vortex. Sonicate con una punta sonicate al 60% de amplitud, añadiendo cinco ciclos de 15 segundos cada uno e intervalos de 15 segundos. Centrifugar a 12.000 g durante cinco minutos.Transfiera 60 microlitros de sobrenadante a una placa de fondo transparente de 96 pocillos y mida la absorbancia del producto sobrenadante a 570 nanómetros. Los resultados del ensayo espectrofotométrico indicaron que 100 nanomolares de PMA indujeron una producción elevada de ROS en comparación con los grupos fMLP y control. Los resultados de la prueba de portaobjetos NBT corroboraron los resultados espectrofotométricos, mostrando a PMA como el estímulo más intenso que induce la producción de ROS en comparación con 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 ...