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

Screening degli inibitori dell'attivazione dell'integrina β2 nei neutrofili umani

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 However, efalizumab was withdrawn due to its lethal side effect - progressive multifocal leukoencephalopathy resulting from JC virus reactivation6,7.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

Autore in primo piano: Sviluppo di un metodo per identificare piccoli antagonisti molecolari dell'attivazione dell'integrina β2 

Una tecnica ad alto rendimento basata sulla citometria a flusso per lo screening di farmaci inibitori delle integrine

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  

Screening ad alto rendimento di composti utilizzando neutrofili isolati da sangue umano in un formato di piastra a 384 pozzetti

Utilizzando una pipetta sierologica, stratificare accuratamente quattro millilitri di sangue umano eparinizzato su otto millilitri di mezzo a gradiente di densità in una provetta da centrifuga da 15 millilitri. Centrifugare il sangue a 550 g per 30-50 minuti a 20 gradi Celsius. Quindi utilizzare una pipetta da un millilitro per rimuovere lo strato superiore contenente plasma e cellule mononucleate.Raccogliere i neutrofili dalla banda torbida inferiore e circa tre o quattro millilitri sotto il liquido limpido in una provetta da centrifuga da 15 millilitri contenente 10 millilitri di PBS. Capovolgere delicatamente il tubo due o tre volte per mescolare la sospensione dei neutrofili. Centrifugare la sospensione di neutrofili a 400G per 10 minuti a 20 gradi Celsius, quindi travasare con cura il surnatante dalla provetta e risospendere il pellet in cinque millilitri di PBS.Anche in questo caso, centrifugare la sospensione a 300 G per 10 minuti a 20 gradi Celsius. Dopo aver rimosso il surnatante, utilizzare l'aspirazione a vuoto per eliminare il surnatante residuo sulla parete del tubo e intorno alla bocca del tubo. Risospendere il pellet in un millilitro di mezzo neutrofilo.Combinare un millilitro di soluzione anticorpale da 1,2 microgrammi per millilitro con 0,2 millilitri di terreno neutrofilo in una provetta da 1,5 millilitri. Aggiungere 25 microlitri di soluzione anticorpale ai pozzetti di controllo negativo in una piastra da 384 pozzetti. In una provetta da 15 millilitri, unire nove millilitri della soluzione anticorpale, 21,6 microlitri della soluzione FMLP e 1,7784 millilitri di mezzo neutrofilo.Aggiungere 25 microlitri di questa miscela ai pozzetti di controllo positivo e di prova in una piastra da 384 pozzetti. Utilizzando una pipetta multicanale, trasferire cinque microlitri di soluzione composta dalla piastra della libreria composta alla piastra da 384 pozzetti. Centrifugare la sospensione a 500G per un minuto.Aggiungere 20 microlitri di sospensione di neutrofili a ciascun pozzetto utilizzando una pipetta a 16 canali. Incubare la piastra su uno shaker a 300 RPM per 10 minuti. Quindi, per fissare le cellule, aggiungere tre microlitri di paraformaldeide al 16% in ciascun pozzetto e incubare su ghiaccio per 10 minuti.

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.