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

Saggi funzionali dei neutrofili per lo screening di diverse vie di segnalazione

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.

Autore in primo piano: Bilanciare velocità, costi e accuratezza nella valutazione della funzione dei neutrofili con il protocollo NeutroFun Screen 

Una serie di tecniche di screening per una rapida panoramica della funzione dei neutrofili

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  

Dosaggio del cloruro blu di nitrotetrazolio (NBT) per la valutazione della produzione di ROS

Inizia sciogliendo 0,0005 grammi di NBT in 10 microlitri di DMSO. Dopo aver volato a vortice per 15 minuti, aggiungere 90 microlitri di soluzione salina bilanciata di Hank o HBSS con calcio e magnesio e un microlitro delle sostanze in esame. Ancora una volta, agitare il tubo per un massimo di due minuti.Per eseguire il test del vetrino NBT, mescolare delicatamente e trasferire due microlitri dei neutrofili polimorfonucleati attivati precedentemente preparati su un vetrino pulito. Incubare il vetrino in camera umidificata a 37 gradi Celsius per 20 minuti. Successivamente, aggiungere un microlitro della soluzione di lavoro NBT sulle celle.Incubare nuovamente per 20 minuti, proteggendo dalla luce. Dopo aver asciugato il vetrino, fissarlo applicando una goccia di metanolo in ogni pozzetto per un minuto. Colorare il vetrino con 0,03 safranina per un altro minuto.In ogni pozzetto, contare 100 cellule casuali, differenziando i neutrofili con e senza depositi di formazano. Per eseguire il saggio di spettrofotometria NBT, miscelare delicatamente e trasferire 90 microlitri dei neutrofili polimorfonucleati attivati precedentemente preparati in una provetta da microcentrifuga pulita. Quindi aggiungere con cura 20 microlitri di una soluzione NBT a sei millimolari e incubare la provetta al buio per 37 gradi Celsius per 20 minuti.Dopo l'incubazione, aggiungere 100 microlitri di SDS al 10% e Vortex. Sonicare con una punta sonicare al 60% di ampiezza, aggiungendo cinque cicli di 15 secondi ciascuno e intervalli di 15 secondi. Centrifugare a 12.000 g per cinque minuti.Trasferire 60 microlitri di surnatante su una piastra a 96 pozzetti con fondo trasparente e misurare l'assorbanza del prodotto surnatante a 570 nanometri. I risultati del test spettrofotometrico hanno indicato che 100 PMA nanomolari hanno indotto un'elevata produzione di ROS rispetto ai gruppi fMLP e di controllo. I risultati del test NBT hanno corroborato i risultati spettrofotometrici, mostrando che il PMA è lo stimolo che induce la produzione di ROS più intenso rispetto all'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 ...