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

인간 호중구에서 β2 Integrin Activation Inhibitors의 스크리닝

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 However, efalizumab was withdrawn due to its lethal side effect - progressive multifocal leukoencephalopathy resulting from JC virus reactivation6,7.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

저자 스포트라이트: β2 Integrin 활성화의 소분자 길항제를 식별하는 방법 개발 

인테그린 억제 약물 스크리닝을 위한 유세포 분석 기반 고처리량 기법

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  

384웰 플레이트 형식의 인간 혈액에서 분리된 호중구를 사용한 화합물의 고처리량 스크리닝

혈청학적 피펫을 사용하여 15밀리리터 원심분리 튜브에 4밀리리터의 헤파린화된 인간 혈액을 8밀리리터의 밀도 구배 배지 위에 조심스럽게 층을 이룹니다. 섭씨 20도에서 30-50분 동안 550G에서 혈액을 원심분리합니다. 그런 다음 1mL 피펫을 사용하여 플라즈마 및 단핵 전지가 포함된 상층을 제거합니다.하부 흐린 띠와 맑은 액체 아래 약 3-4밀리리터의 호중구를 10밀리리터의 PBS가 들어 있는 15밀리리터의 원심분리 튜브에 모읍니다. 튜브를 부드럽게 뒤집어 호중구 현탁액을 혼합합니다. 호중구 현탁액을 섭씨 20도에서 400G에서 10분 동안 원심분리한 다음 튜브에서 상층액을 조심스럽게 디캔팅하고 펠릿을 5밀리리터의 PBS에 재현탁시킵니다.다시 섭씨 300도에서 10분 동안 서스펜션을 20G로 원심분리합니다. 상층액을 제거한 후 진공 흡입을 사용하여 튜브 벽과 튜브 입구 주변에 남아 있는 상층액을 제거합니다. 펠릿을 1밀리리터의 호중구 배지에 재현탁시킵니다.1.5 밀리리터 튜브에 1 밀리리터당 1.2 마이크로그램의 항체 용액과 0.2 밀리리터의 호중구 배지를 결합합니다. 25 마이크로리터의 항체 용액을 384웰 플레이트의 음성 대조군 웰에 추가합니다. 15밀리리터 튜브에 9밀리리터의 항체 용액, 21.6마이크로리터의 FMLP 용액 및 1.7784밀리리터의 호중구 배지를 결합합니다.이 혼합물 25마이크로리터를 양성 대조군에 추가하고 384웰 플레이트에서 웰을 테스트합니다. 다중 채널 피펫을 사용하여 5마이크로리터의 복합 용액을 화합물 라이브러리 플레이트에서 384웰 플레이트로 옮깁니다. 서스펜션을 500G에서 1분 동안 원심분리합니다.16채널 피펫을 사용하여 각 웰에 20마이크로리터의 호중구 현탁액을 추가합니다. 접시를 300RPM의 셰이커에서 10분 동안 배양합니다. 그런 다음 세포를 고정하기 위해 각 웰에 16% 파라포름알데히드 3마이크로리터를 넣고 얼음 위에서 10분 동안 배양합니다.

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.