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

Criblage d’inhibiteurs de l’activation de l’intégrine β2 dans les neutrophiles humains

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 However, efalizumab was withdrawn due to its lethal side effect - progressive multifocal leukoencephalopathy resulting from JC virus reactivation6,7.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.

Pleins feux sur l’auteur : Développement d’une méthode d’identification des petits antagonistes moléculaires de l’activation de l’intégrine β2 

Technique à haut débit basée sur la cytométrie en flux pour le criblage de médicaments inhibiteurs de l’intégrine

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  

Criblage à haut débit de composés à l’aide de neutrophiles isolés de sang humain dans un format de plaque à 384 puits

À l’aide d’une pipette sérologique, superposer soigneusement quatre millilitres de sang humain hépariné sur huit millilitres de milieu à gradient de densité dans un tube à centrifuger de 15 millilitres. Centrifuger le sang à 550G pendant 30 à 50 minutes à 20 degrés Celsius. Ensuite, utilisez une pipette d’un millilitre pour retirer la couche supérieure contenant le plasma et les cellules mononucléées.Recueillir les neutrophiles de la bande trouble inférieure et à environ trois à quatre millilitres sous le liquide clair dans un tube à centrifuger de 15 millilitres contenant 10 millilitres de PBS. Retournez doucement le tube deux à trois fois pour mélanger la suspension de neutrophiles. Centrifugez la suspension de neutrophiles à 400 G pendant 10 minutes à 20 degrés Celsius, puis décantez soigneusement le surnageant du tube et remettez la pastille en suspension dans cinq millilitres de PBS.Encore une fois, centrifugez la suspension à 300G pendant 10 minutes à 20 degrés Celsius. Après avoir retiré le surnageant, utilisez l’aspiration sous vide pour éliminer le surnageant résiduel sur la paroi du tube et autour de l’embouchure du tube. Remettre la pastille en suspension dans un millilitre de milieu neutrophile.Combinez un millilitre de solution d’anticorps de 1,2 microgramme par millilitre avec 0,2 millilitre de milieu neutrophile dans un tube de 1,5 millilitre. Ajouter 25 microlitres de solution d’anticorps dans les puits témoins négatifs dans une plaque à 384 puits. Dans un tube de 15 millilitres, mélanger neuf millilitres de solution d’anticorps, 21,6 microlitres de solution FMLP et 1,7784 millilitre de milieu neutrophile.Ajouter 25 microlitres de ce mélange aux puits de contrôle positif et d’essai dans une plaque de 384 puits. À l’aide d’une pipette multicanaux, transférez cinq microlitres de solution composée de la plaque de la bibliothèque de composés à la plaque de 384 puits. Centrifugez la suspension à 500G pendant une minute.Ajouter 20 microlitres de suspension de neutrophiles dans chaque puits à l’aide d’une pipette à 16 canaux. Incuber l’assiette sur un shaker à 300 tr/min pendant 10 minutes. Ensuite, pour fixer les cellules, ajoutez trois microlitres de 16% de paraformaldéhyde dans chaque puits, et incubez sur de la glace pendant 10 minutes.

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.