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

Cribado de inhibidores de la activación de integrinas β2 en neutrófilos humanos

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 However, efalizumab was withdrawn due to its lethal side effect - progressive multifocal leukoencephalopathy resulting from JC virus reactivation6,7.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

Autor destacado: Desarrollo de un método para identificar pequeños antagonistas moleculares de la activación de la integrina β2 

Una técnica de alto rendimiento basada en citometría de flujo para el cribado de fármacos inhibidores de integrinas

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  

Cribado de alto rendimiento de compuestos utilizando neutrófilos aislados de sangre humana en un formato de placa de 384 pocillos

Con una pipeta serológica, coloque cuidadosamente cuatro mililitros de sangre humana heparinizada sobre ocho mililitros de medio de gradiente de densidad en un tubo de centrífuga de 15 mililitros. Centrifugar la sangre a 550 g durante 30 a 50 minutos a 20 grados centígrados. A continuación, utilice una pipeta de un mililitro para eliminar la capa superior que contiene plasma y células mononucleares.Recoja los neutrófilos de la banda turbia inferior y aproximadamente de tres a cuatro mililitros por debajo del líquido transparente en un tubo de centrífuga de 15 mililitros que contenga 10 mililitros de PBS. Invierta suavemente el tubo dos o tres veces para mezclar la suspensión de neutrófilos. Centrifugar la suspensión de neutrófilos a 400G durante 10 minutos a 20 grados centígrados, luego decantar cuidadosamente el sobrenadante del tubo y volver a suspender el gránulo en cinco mililitros de PBS.De nuevo, centrifuga la suspensión a 300G durante 10 minutos a 20 grados centígrados. Después de quitar el sobrenadante, use succión al vacío para eliminar el sobrenadante residual en la pared del tubo y alrededor de la boca del tubo. Resuspenda el pellet en un mililitro de medio neutrófilo.Combine un mililitro de solución de anticuerpos de 1,2 microgramos por mililitro con 0,2 mililitros de medio neutrófilo en un tubo de 1,5 mililitros. Agregue 25 microlitros de solución de anticuerpos a los pocillos de control negativo en una placa de 384 pocillos. En un tubo de 15 mililitros, combine nueve mililitros de la solución de anticuerpos, 21,6 microlitros de la solución FMLP y 1,7784 mililitros de medio neutrófilo.Agregue 25 microlitros de esta mezcla al control positivo y pruebe los pocillos en una placa de 384 pocillos. Con una pipeta multicanal, transfiera cinco microlitros de solución compuesta de la placa de biblioteca de compuestos a la placa de 384 pocillos. Centrifugar la suspensión a 500 G durante un minuto.Agregue 20 microlitros de suspensión de neutrófilos a cada pocillo con una pipeta de 16 canales. Incubar la placa en una coctelera a 300 RPM durante 10 minutos. Luego, para fijar las células, agregue tres microlitros de paraformaldehído al 16% a cada pocillo e incube en hielo durante 10 minutos.

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.