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

Triagem de inibidores de ativação da integrina β2 em 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 However, efalizumab was withdrawn due to its lethal side effect - progressive multifocal leukoencephalopathy resulting from JC virus reactivation6,7.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 em destaque: Desenvolvimento de um método para identificar pequenos antagonistas moleculares da ativação da integrina β2 

Uma Técnica de Alto Rendimento Baseada em Citometria de Fluxo para Triagem de Drogas Inibidoras 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  

Triagem de alto rendimento de compostos usando neutrófilos isolados do sangue humano em um formato de placa de 384 poços

Usando uma pipeta sorológica, coloque cuidadosamente quatro mililitros de sangue humano heparinizado sobre oito mililitros de meio de gradiente de densidade em um tubo centrífugo de 15 mililitros. Centrifugar o sangue a 550G durante 30 a 50 minutos a 20 graus Celsius. Em seguida, use uma pipeta de um mililitro para remover a camada superior contendo plasma e células mononucleares.Coletar neutrófilos da faixa turva inferior e aproximadamente três a quatro mililitros abaixo do líquido claro em um tubo de centrífuga de 15 mililitros contendo 10 mililitros de PBS. Inverta suavemente o tubo duas a três vezes para misturar a suspensão de neutrófilos. Centrifugar a suspensão de neutrófilos a 400G por 10 minutos a 20 graus Celsius, depois decantar cuidadosamente o sobrenadante do tubo e ressuspender o pellet em cinco mililitros de PBS.Novamente, centrifugar a suspensão a 300G por 10 minutos a 20 graus Celsius. Depois de remover o sobrenadante, use sucção a vácuo para eliminar o sobrenadante residual na parede do tubo e ao redor da boca do tubo. Ressuspender o pellet em um mililitro de meio neutrófilo.Combine um mililitro de solução de anticorpos de 1,2 microgramas por mililitro com 0,2 mililitros de meio neutrófilo em um tubo de 1,5 mililitro. Adicionar 25 microlitros de solução de anticorpos aos poços de controle negativo em uma placa de 384 poços. Em um tubo de 15 mililitros, combinar nove mililitros da solução de anticorpos, 21,6 microlitros da solução FMLP e 1,7784 mililitros de meio neutrófilo.Adicione 25 microlitros dessa mistura aos poços de controle positivo e teste em uma placa de 384 poços. Usando uma pipeta multicanal, transfira cinco microlitros de solução composta da placa de biblioteca composta para a placa de 384 poços. Centrifugar a suspensão a 500G por um minuto.Adicione 20 microlitros de suspensão de neutrófilos a cada poço usando uma pipeta de 16 canais. Incubar a placa em um agitador a 300 RPM por 10 minutos. Em seguida, para fixar as células, adicione três microlitros de paraformaldeído a 16% em cada poço e incube no gelo por 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.