neutrophil phagocytosis assay

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 Neutrophil Phagocytosis Assay at JoVE.com

Ensaio de Fagocitose de Neutrófilos

Adicione aproximadamente 0,75 miligramas de levedura seca a 200 microlitros de Hanks'Balanced Salt Solution, ou HBSS, contendo íons cálcio e magnésio. Em seguida, incube a mistura em um ThermoMixer a 100 graus Celsius e 500 rpm por um mínimo de 15 minutos. Após a incubação, transfira cinco microlitros de suspensão de levedura para 45 microlitros de corante azul de tripano a 0,2%.Conte as células de levedura usando uma câmara de Neubauer. Ajustar a concentração da suspensão inicial para 33.000 células de levedura por microlitro usando HBSS com cálcio e magnésio. Em seguida, misture e transfira suavemente cinco microlitros dos neutrófilos polimorfonucleares ativados previamente preparados para cinco microlitros da suspensão de células de levedura em um novo tubo de microcentrífuga estéril.Transfira imediatamente seis microlitros dos polimorfonucleares neutrófilos e da suspensão de levedura em três poços de uma lâmina de vidro limpa, dois microlitros cada, e incube a lâmina em uma câmara umidificada por 40 minutos. Após coloração com panóptico rápido, observar as células ao microscópio. Embora tanto a FMLP de 100 nanomolares quanto o peptídeo antimicromolar de 16 micromolares tenham induzido um aumento do engolfamento de leveduras, a diferença não foi estatisticamente significativa até a dupla estimulação, que aumentou significativamente a fagocitose em comparação com o grupo controle.

Research

JoVE Journal

Immunology and Infection

Neutrophil Phagocytosis Assay

222 visualizações.  University of Brasilia. Adicione aproximadamente 0,75 miligramas de levedura seca a 200 microlitros de Hanks'Balanced Salt Solution, ou HBSS, contendo íons cálcio e magnésio. Em seguida, incube a mistura em um ThermoMixer a 100 graus Celsius e 500 rpm por um mínimo de 15 minutos. Após a incubação, transfira cinco microlitros de suspensão de levedura para 45 microlitros de corante azul de tripano a 0,2%.Conte as células de levedura usando uma câmara de Neubauer. Ajustar a concentração da suspensão...

Vídeo: Ensaio de Fagocitose de Neutrófilos

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 chemotaxis, reverse migration, phagocytosis, degranulation of cytotoxic enzymes and metabolites, and release of DNA as neutrophil extracellular traps (NETs). Neutrophils not only have tightly regulated signaling themselves, but also participate in the regulation of other components of the immune system. As fresh neutrophils are terminally differentiated, short-lived, and highly variable among individuals, it is important to make the most of the collected samples. Researchers often need to perform screening assays to assess an overview of the many neutrophil functions that may be affected by specific conditions under evaluation. A set of tests following a single isolation process of normal density neutrophils was developed to address this need, seeking a balance between speed, comprehensiveness, cost, and accuracy. The results can be used to reason and guide in-depth follow-up studies. This procedure can be carried out in an average time of 4 h and includes the evaluation of cell viability, reactive oxygen species (ROS) production, real-time migration, and phagocytosis of yeast on glass slides, leaving enough cells for more detailed approaches like omics studies. Moreover, the procedure includes a way to easily observe a preliminary suggestion of NETs after fast panoptic staining observed by light microscopy, with a lack of specific markers, albeit enough to indicate if further efforts in that way would be worthwhile. The diversity of functions tested combines common points among tests, reducing the analysis time and expenses. The procedure was named NeutroFun Screen, and although having&#160;limitations, it balances the aforementioned factors. Furthermore, the aim of this work is not a definite test set, but rather a guideline that can easily be adjusted to each lab&#39;s resources and demands.

This protocol features a set of neutrophil functional assays to be used as a screening method to cover functions from different signaling pathways. The protocol includes an initial and simple evaluation of cell viability, purity, reactive oxygen species production, real-time migration, phagocytosis, and a preliminary suggestion of neutrophil extracellular traps.

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 ...