Name: High-Throughput Screening of Compounds Using Neutrophils Isolated from Human Blood in a 384-Well Plate Format
Uploaded: 2024-02-02T12:00:00
Description: Watch this Scientific Journal Video about High-Throughput Screening of Compounds Using Neutrophils Isolated from Human Blood in a 384-Well Plate Format at JoVE.com

high-throughput screening of compounds using neutrophils isolated from human blood in a 384-well plate format

İnsan Nötrofillerinde β2 İntegrin Aktivasyon İnhibitörlerinin Taranması

Many inflammatory diseases are characterized by the infiltration of neutrophils at the site of swelling or injury1. To infiltrate these tissues, neutrophils must complete the neutrophil recruitment cascade, which involves arrest to the endothelium, extravasation across the vessel wall, and recruitment into the tissue2. Circulating neutrophils need &#946;2 integrin activation to complete this cascade, especially for the arrest phase. Thus, integrin-inhibiting drugs that reduce neutrophil adhesion, extravasation, and recruitment may effectively treat inflammatory diseases3,4.
&#946;2 integrins have been targeted for inflammatory diseases before. Efalizumab, a monoclonal antibody directly targeting integrin &#945;L&#946;2, was developed to treat psoriasis5. However, efalizumab was withdrawn due to its lethal side effect - progressive multifocal leukoencephalopathy resulting from JC virus reactivation6,7. New anti-inflammatory integrin-based therapies should consider maintaining the anti-infection functions of leukocytes to minimize side effects. The side effects of efalizumab might be due to the prolonged circulation of monoclonal antibodies in the bloodstream, which could inhibit immune functions in the long term8. A recent study shows that efalizumab mediates &#945;L&#946;2 crosslinking and the unwanted internalization of &#945;4 integrins, providing an alternative explanation for the side effects9. Thus, short-lived, small-molecule antagonists might avoid this problem.
A high-throughput method to screen small-molecule &#946;2 integrin antagonists using human neutrophils is presented here. &#946;2 integrin activation requires conformational changes of the integrin ectodomain to gain access to and increase its binding affinity to its ligand. In the canonical switchblade model, the bent-closed integrin ectodomain first extends to an extended-closed conformation and then opens its headpiece to a fully activated extended-open conformation10,11,12,13. There is also an alternative pathway that starts from the bent-closed to bent-open and extended-open, eventually14,15,16,17,18,19. The conformation-specific antibody mAb24 binds to an epitope in the human &#946;2-I-like domain when the headpiece of the ectodomain is open20,21,22,23.
Here, mAb24-APC is used to determine whether the &#946;2 integrins are activated. To activate neutrophils and integrin, N-formylmethionyl-leucyl-phenylalanine (fMLP), a bacterial-derived short chemotactic peptide that can activate neutrophil &#946;2 integrins24, is used as a stimulus in this protocol. When fMLP binds to the Fpr1 on neutrophils, downstream signaling cascades involving G-proteins, phospholipase C&#946;, and phosphoinositide 3-kinase &#947; are activated. These signaling events ultimately result in integrin activation via the inside-out signaling pathway18,25. Besides small molecule antagonists that directly bind to &#946;2 integrins and prevent conformational changes of integrin activation26, compounds that can inhibit components in the &#946;2 integrin inside-out activation signaling pathway would also be detected with this method. Automated flow cytometers enable high-throughput screening. Identifying new antagonists may not only deepen our understanding of integrin physiology but also provide translational insight into integrin-based anti-inflammation therapy.

Yazar Spotlight: β2 integrin aktivasyonunun küçük moleküler antagonistlerini tanımlamak için bir yöntemin geliştirilmesi 

İntegrin İnhibitör İlaçların Taranması için Flow Sitometri Tabanlı Yüksek Verimli Bir Teknik

Our research is focused on neutrophil integrin, and in this study we aim to establish a method to find small molecular antagonists of Beta-2 integrin activation. We believe with this method we can find new antagonists that may deepen our understanding in integrin physiology, and also provide translational insight into integrin-based anti-inflammatory therapy. In the future, we will focus on discovering new anti-inflammatory drugs and dissecting their mechanisms of action.

Watch this Scientific Journal Video about High-Throughput Screening of Compounds Using Neutrophils Isolated from Human Blood in a 384-Well Plate Format at JoVE.com

High-Throughput Screening of Compounds Using Neutrophils Isolated from Human Blood in a 384-Well Plate Format  

384 Kuyulu Plaka Formatında İnsan Kanından İzole Edilen Nötrofiller Kullanılarak Bileşiklerin Yüksek Verimli Taranması

Serolojik bir pipet kullanarak, 15 mililitrelik bir santrifüj tüpünde sekiz mililitre yoğunluk gradyan ortamı üzerine dört mililitre heparinize insan kanını dikkatlice katmanlayın. Kanı 550G'de 20 santigrat derecede 30 ila 50 dakika santrifüjleyin. Ardından, plazma ve mononükleer hücreler içeren üst tabakayı çıkarmak için bir mililitrelik bir pipet kullanın.Alt bulutlu banttan ve berrak sıvının yaklaşık üç ila dört mililitre altındaki nötrofilleri, 10 mililitre PBS içeren 15 mililitrelik bir santrifüj tüpünde toplayın. Nötrofil süspansiyonunu karıştırmak için tüpü iki ila üç kez hafifçe ters çevirin. Nötrofil süspansiyonunu 400G'de 20 santigrat derecede 10 dakika santrifüjleyin, ardından süpernatanı tüpten dikkatlice boşaltın ve peleti beş mililitre PBS'de yeniden süspanse edin.Yine, süspansiyonu 300G'de 20 santigrat derecede 10 dakika santrifüjleyin. Süpernatanı çıkardıktan sonra, tüp duvarında ve tüp ağzının çevresinde kalan süpernatanı ortadan kaldırmak için vakumlu emme kullanın. Peletin bir mililitre nötrofil ortamında yeniden süspanse edilmesi.Mililitre antikor çözeltisi başına bir mililitre 1.2 mikrogramı, 1.5 mililitrelik bir tüpte 0.2 mililitre nötrofil ortamı ile birleştirin. 384 oyuklu bir plakadaki negatif kontrol kuyucuklarına 25 mikrolitre antikor çözeltisi ekleyin. 15 mililitrelik bir tüpte, dokuz mililitre antikor çözeltisini, 21.6 mikrolitre FMLP çözeltisini ve 1.7784 mililitre nötrofil ortamını birleştirin.Bu karışımdan 25 mikrolitreyi pozitif kontrole ekleyin ve 384 oyuklu bir plakada kuyucukları test edin. Çok kanallı bir pipet kullanarak, beş mikrolitre bileşik çözeltiyi bileşik kitaplık plakasından 384 oyuklu plakaya aktarın. Süspansiyonu 500G'de bir dakika santrifüjleyin.16 kanallı bir pipet kullanarak her oyuğa 20 mikrolitre nötrofil süspansiyonu ekleyin. Plakayı bir çalkalayıcı üzerinde 300 RPM'de 10 dakika inkübe edin. Daha sonra hücreleri sabitlemek için, her bir oyuğa üç mikrolitre% 16 paraformaldehit ekleyin ve 10 dakika boyunca buz üzerinde inkübe edin.

high-throughput-screening-compounds-using-neutrophils-isolated-from

Research

JoVE Journal

Immunology and Infection

High-Throughput Screening of Compounds Using Neutrophils Isolated from Human Blood in a 384-Well Plate Format

A Flow Cytometry-Based High-Throughput Technique for Screening Integrin-Inhibitory Drugs

This protocol aims to establish a method for identifying small molecular antagonists of &#946;2 integrin activation, utilizing conformational-change-reporting antibodies and high-throughput flow cytometry. The method can also serve as a guide for other antibody-based high-throughput screening methods. &#946;2 integrins are leukocyte-specific adhesion molecules that are crucial in immune responses. Neutrophils rely on integrin activation to exit the bloodstream, not only to fight infections but also to be involved in multiple inflammatory diseases. Controlling &#946;2 integrin activation presents a viable approach for treating neutrophil-associated inflammatory diseases. In this protocol, a monoclonal antibody, mAb24, which specifically binds to the high-affinity headpiece of &#946;2 integrins, is utilized to quantify &#946;2 integrin activation on isolated primary human neutrophils. N-formylmethionyl-leucyl-phenylalanine (fMLP) is used as a stimulus to activate neutrophil &#946;2 integrins. A high-throughput flow cytometer capable of automatically running 384-well plate samples was used in this study. The effects of 320 chemicals on &#946;2 integrin inhibition are assessed within 3 h. Molecules that directly target &#946;2 integrins or target molecules in the G protein-coupled receptor-initiated integrin inside-out activation signaling pathway can be identified through this approach.

This protocol describes a flow cytometry-based, high-throughput screening method to identify small-molecule drugs that inhibit &#946;2 integrin activation on human neutrophils.