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

Nøytrofile funksjonelle analyser for screening av ulike signalveier

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.

Forfatter Spotlight: Balansering av hastighet, kostnad og nøyaktighet i nøytrofil funksjonsvurdering med NeutroFun Screen Protocol 

Et sett med screeningteknikker for en rask oversikt over nøytrofilfunksjonen

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  

Nitrotetrazoliumblåklorid (NBT)-analyse for evaluering av ROS-produksjon

Begynn med å oppløse 0,0005 gram NBT i 10 mikroliter DMSO. Etter vortexing i 15 minutter, tilsett 90 mikroliter Hank's Balanced Salt Solution eller HBSS med kalsium og magnesium og en mikroliter av teststoffene. Igjen, virvel røret i opptil to minutter.For å utføre NBT-lysbildetesten, bland og overfør forsiktig to mikroliter av de tidligere tilberedte aktiverte polymorfonukleære nøytrofilene til et rent glassglass. Inkuber lysbildet i fuktet kammer ved 37 grader Celsius i 20 minutter. Deretter legger du til en mikroliter av NBT-arbeidsløsningen over cellene.Inkuber igjen i 20 minutter, beskytter mot lys. Etter tørking av lysbildet, fiks det ved å påføre en dråpe metanol i hver brønn i ett minutt. Beis lysbildet med 0,03 safranin i ytterligere ett minutt.I hver brønn, telle 100 tilfeldige celler, skille nøytrofiler med og uten formazan innskudd. For å utføre NBT-spektrofotometrianalysen, bland og overfør forsiktig 90 mikroliter av de tidligere tilberedte aktiverte polymorfonukleære nøytrofilene til et rent mikrosentrifugerør. Deretter tilsettes forsiktig 20 mikroliter av en seks millimolar NBT-løsning og inkuber røret i mørket i 37 grader Celsius i 20 minutter.Etter inkubasjon, tilsett 100 mikroliter 10% SDS og Vortex. Sonikere med et spisssonikat ved 60% amplitude, og legg til fem sykluser på 15 sekunder hver og 15 sekunders intervaller. Sentrifuge ved 12.000g i fem minutter.Overfør 60 mikroliter supernatant til en klar bunnplate med 96 brønner og mål absorbansen til supernatantproduktet ved 570 nanometer. Resultatene av den spektrofotometriske analysen indikerte at 100 nanomolar PMA induserte forhøyet ROS-produksjon sammenlignet med fMLP og kontrollgruppene. NBT-lysbildetestresultatene bekreftet de spektrofotometriske resultatene, og viste PMA som den mest intense ROS-produksjonsinduserende stimulansen sammenlignet med 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 ...