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

Neutrofiele functionele assays voor het screenen van verschillende signaalroutes

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.

Auteur in de schijnwerpers: Balanceren tussen snelheid, kosten en nauwkeurigheid bij de beoordeling van de functie van neutrofielen met het NeutroFun-schermprotocol 

Een reeks screeningstechnieken voor een snel overzicht van de neutrofielenfunctie

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  

Nitrotetrazolium Blue Chloride (NBT)-test voor het evalueren van ROS-productie

Begin met het oplossen van 0,0005 gram NBT in 10 microliter DMSO. Voeg na 15 minuten vortexen 90 microliter Hank's Balanced Salt Solution of HBSS met calcium en magnesium en één microliter van de teststoffen toe. Nogmaals, draai de buis maximaal twee minuten in een draaikolf.Om de NBT-objectglaasjestest uit te voeren, mengt u voorzichtig twee microliter van de eerder bereide geactiveerde polymorfonucleaire neutrofielen en brengt u deze over op een schoon glasplaatje. Incubeer het objectglaasje in een bevochtigde kamer bij 37 graden Celsius gedurende 20 minuten. Voeg vervolgens een microliter van de NBT-werkoplossing toe aan de cellen.Incubeer opnieuw gedurende 20 minuten, ter bescherming tegen licht. Nadat u het glaasje hebt gedroogd, fixeert u het door gedurende één minuut een druppel methanol in elk putje aan te brengen. Kleur het glaasje nog een minuut met 0,03 safranine.Tel in elk putje 100 willekeurige cellen, waarbij neutrofielen met en zonder formazan-afzettingen worden onderscheiden. Om de NBT-spectrofotometrietest uit te voeren, mengt u voorzichtig 90 microliter van de eerder bereide geactiveerde polymorfonucleaire neutrofielen en brengt u deze over naar een schone microcentrifugebuis. Voeg vervolgens voorzichtig 20 microliter van een NBT-oplossing van zes millimolaire en incubeer de buis gedurende 20 minuten in het donker gedurende 37 graden Celsius.Voeg na incubatie 100 microliter 10% SDS en Vortex toe. Sonicate met een tip sonicate op 60%amplitude, waarbij vijf cycli van elk 15 seconden en intervallen van 15 seconden worden toegevoegd. Centrifugeer op 12.000 g gedurende vijf minuten.Breng 60 microliter supernatans over naar een doorzichtige bodemplaat met 96 putjes en meet de absorptie van het supernatans product op 570 nanometer. De resultaten van de spectrofotometrische test gaven aan dat 100 nanomolaire PMA een verhoogde ROS-productie induceerde in vergelijking met de fMLP- en controlegroepen. De resultaten van de NBT-objectglaasjestest bevestigden de spectrofotometrische resultaten en toonden aan dat PMA de meest intense ROS-productie-inducerende stimulus was in vergelijking met 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 ...