Name: 호중구 식세포증 분석
Uploaded: 2024-02-09T12:40:00
Description: Watch this Scientific Journal Video about Neutrophil Phagocytosis Assay at JoVE.com

neutrophil phagocytosis assay

다양한 신호전달 경로를 스크리닝하기 위한 호중구 기능 분석

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.

저자 스포트라이트: NeutroFun Screen Protocol을 사용한 호중구 기능 평가의 속도, 비용 및 정확성 균형 맞추기 

호중구 기능에 대한 간략한 개요를 위한 일련의 스크리닝 기법

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

호중구 식세포증 분석

칼슘과 마그네슘 이온이 함유된 200마이크로리터의 Hanks'Balanced Salt Solution(HBSS)에 약 0.75mg의 건조 효모를 첨가합니다. 다음으로, 혼합물을 섭씨 100도 및 500rpm의 ThermoMixer에서 최소 15분 동안 배양합니다. 배양 후 5 마이크로 리터의 효모 현탁액을 0.2 % 트리판 블루 염료의 45 마이크로 리터로 옮깁니다.Neubauer 챔버를 사용하여 효모 세포를 계산합니다. 처음 현탁액의 농도를 33, 칼슘과 마그네슘을 가진 HBSS를 사용하여 마이크로리터 당 000의 효모 세포에 조정하십시오. 다음으로, 이전에 준비한 활성화된 다형핵 호중구 5마이크로리터를 새로운 멸균 미세원심분리 튜브에서 5마이크로리터의 효모세포 현탁액으로 부드럽게 혼합하고 옮깁니다.즉시 6 마이크로 리터의 다형 핵 호중구와 효모 현탁액을 각각 2 마이크로 리터의 깨끗한 유리 슬라이드의 3 개의 웰로 옮기고 40 분 동안 가습 챔버에서 슬라이드를 배양합니다. 빠른 파놉틱으로 염색한 후 현미경으로 세포를 관찰합니다. 100나노몰 FMLP와 16마이크로몰 항균 펩타이드 모두 효모 삼킴 증가를 유도했지만, 대조군에 비해 식세포작용을 크게 향상시킨 이중 자극 전까지는 그 차이가 통계적으로 유의하지 않았습니다.

Research

JoVE Journal

Immunology and Infection

Neutrophil Phagocytosis Assay

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 ...