Name: Real-Time Polymorphonuclear Neutrophils (PMN) Chemotaxis Assay
Uploaded: 2024-02-09T12:40:00
Description: Watch this Scientific Journal Video about Real-Time Polymorphonuclear Neutrophils PMN Chemotaxis Assay at JoVE.com

real-time polymorphonuclear neutrophils (pmn) chemotaxis assay

用于筛选不同信号通路的中性粒细胞功能检测

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.

作者聚焦：使用 NeutroFun 筛选方案平衡中性粒细胞功能评估的速度、成本和准确性 

一套快速了解中性粒细胞功能的筛查技术

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 Real-Time Polymorphonuclear Neutrophils PMN Chemotaxis Assay at JoVE.com

Real-Time Polymorphonuclear Neutrophils (PMN) Chemotaxis Assay  

实时荧光定量多形核中性粒细胞 （PMN） 趋化性测定

通过向基于阻抗的实时细胞分析仪或RTCA板的下腔室中加入160微升化学引诱剂来制备趋化梯度。对于阴性对照和空白，加入 160 微升含钙和镁的 Hanks'Balanced 盐溶液或 HBSS。连接上腔室并加入 25 微升含有钙和镁的 HBSS。在室温下孵育至少一小时以形成趋化梯度。接下来，轻轻地混合并转移60微升先前制备的活化多形核中性粒细胞进入上腔室。然后在空白中加入 60 微升含钙和镁的 HBSS。将RTCA板放入分析仪中，并对其软件进行编程，以每60秒测量一次细胞指数，持续两小时。用获得的数据拟合曲线。实时细胞迁移对中性粒细胞表现出 100 纳摩尔 fMLP 的趋化作用。

Research

JoVE Journal

Immunology and Infection

Real-Time Polymorphonuclear Neutrophils (PMN) Chemotaxis 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 ...