neutrophil isolation and activation

Farklı Sinyal Yollarının Tizlenmesi için Nötrofil Fonksiyonel Tahliller

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.

Yazar Spotlight: NeutroFun Ekran Protokolü ile Nötrofil Fonksiyon Değerlendirmesinde Hız, Maliyet ve Doğruluğun Dengelenmesi 

Nötrofil fonksiyonuna hızlı bir genel bakış için bir dizi tarama tekniği

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 Isolation and Activation at JoVE.com

Nötrofil İzolasyonu ve Aktivasyonu

50 mililitrelik konik tüplerde %60 ve %70 ayırma ortamının 12 mililitre seyreltilmesini hazırlayın. Ardından, beş mililitrelik bir pipet kullanarak %70 seyreltme üzerine %60 seyreltme seferinde dört mililitre ekleyerek gradyanı aşağıdan yukarıya hazırlayın. Daha sonra 12 mililitre heparinize kanı yoğunluk gradyanının üzerine dikkatlice yerleştirin ve oda sıcaklığında 15 dakika boyunca 200G'de santrifüjleyin.Plazma ve mononükleer hücre katmanlarını atın. Daha sonra eritrosit peletinin üzerindeki tabakanın yaklaşık 7,5 mililitresini dikkatlice iki adet 15 mililitrelik konik tüpe aktarın. Tüpleri 300G'de 19 santigrat derecede beş dakika santrifüjleyin.Hücre peletini Hanks'in Dengeli Tuz Çözeltisi veya HBSS ile yıkamaya devam edin. Sonra hipotonik lizis yapın ve kalan kırmızı kan hücrelerini çıkarın. Süpernatanı attıktan sonra, kalan tampondaki polimorfonükleer nötrofilleri yavaşça yeniden süspanse edin ve buz gibi soğuk bir mikro santrifüj tüpüne aktarın.Daha sonra, hücre süspansiyonunun üç adet bir mikrolitrelik alikotunu temiz bir cam slayta aktarın. Hücre morfolojisini ve saflığını değerlendirmek için hızlı panoptik ile boyayın. Hücreleri mikroskop altında gözlemleyin ve her kuyucukta 300 rastgele hücre sayın.Ayırmanın saflığını değerlendirmek için nötrofilleri ve diğer hücre tiplerini işaretleyin. Daha sonra, hücre süspansiyonunun bir mikrolitresini 49 mikrolitre% 0.2 tripan mavisi boyaya aktarın ve bir Neubauer odası kullanarak ölü ve canlı hücreleri sayın. Kalsiyum ve magnezyum ile desteklenmiş% 50 otolog plazma ve% 50 HBSS ile hücre konsantrasyonunu mikrolitre başına 6.667 hücreye ayarlayın.Daha sonra hücre süspansiyonunu, negatif kontrol de dahil olmak üzere test koşullarına karşılık gelen 1.5 mililitrelik mikro santrifüj tüpleri arasında eşit olarak bölün. Her koşul için bir aktivasyon sistemi hazırlayın ve nihai hücre konsantrasyonunun mikrolitre başına 6.600 polimorfonükleer nötrofilde kalmasını sağlayın. Dönmeden 37 santigrat derecede inkübe edin.

Research

JoVE Journal

Immunology and Infection

Neutrophil Isolation and Activation

288 Görüntüleme.  University of Brasilia. 50 mililitrelik konik tüplerde %60 ve %70 ayırma ortamının 12 mililitre seyreltilmesini hazırlayın. Ardından, beş mililitrelik bir pipet kullanarak %70 seyreltme üzerine %60 seyreltme seferinde dört mililitre ekleyerek gradyanı aşağıdan yukarıya hazırlayın. Daha sonra 12 mililitre heparinize kanı yoğunluk gradyanının üzerine dikkatlice yerleştirin ve oda sıcaklığında 15 dakika boyunca 200G'de santrifüjleyin.Plazma ve mononükleer hücre katmanlarını atın....

Video: Nötrofil İzolasyonu ve Aktivasyonu

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 ...