Name: Высокопроизводительный скрининг соединений с использованием нейтрофилов, выделенных из крови человека, в формате 384-луночного планшета
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

Скрининг ингибиторов активации β2 интегрина в нейтрофилах человека

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 However, efalizumab was withdrawn due to its lethal side effect - progressive multifocal leukoencephalopathy resulting from JC virus reactivation6,7.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

В центре внимания автора: Разработка метода идентификации низкомолекулярных антагонистов активации β2-интегрина 

Высокопроизводительный метод скрининга интегрин-ингибирующих препаратов на основе проточной цитометрии

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

Высокопроизводительный скрининг соединений с использованием нейтрофилов, выделенных из крови человека, в формате 384-луночного планшета  

С помощью серологической пипетки осторожно нанесите четыре миллилитра гепаринизированной человеческой крови на восемь миллилитров среды градиента плотности в центрифужную пробирку объемом 15 миллилитров. Центрифугируйте кровь при 550 г в течение 30-50 минут при температуре 20 градусов Цельсия. Затем с помощью пипетки объемом один миллилитр удалите верхний слой, содержащий плазму и мононуклеарные клетки.Соберите нейтрофилы из нижней мутной полосы и примерно на три-четыре миллилитра ниже прозрачной жидкости в 15-миллилитровую центрифужную пробирку, содержащую 10 миллилитров PBS. Аккуратно переверните пробирку два-три раза, чтобы смешать суспензию нейтрофилов. Центрифугируйте суспензию нейтрофилов при 400G в течение 10 минут при температуре 20 градусов Цельсия, затем осторожно сцедите надосадочную жидкость из пробирки и повторно суспендируйте гранулу в пяти миллилитрах PBS.Снова центрифугируйте суспензию при 300G в течение 10 минут при 20 градусах Цельсия. После удаления надосадочной жидкости используйте вакуумное отсасывание, чтобы удалить остаточную надосадочную жидкость на стенках трубки и вокруг устья трубки. Ресуспендируйте гранулу в одном миллилитре нейтрофильной среды.Соедините один миллилитр раствора антител 1,2 мкг на миллилитр с 0,2 миллилитра нейтрофильной среды в пробирке объемом 1,5 миллилитра. Добавьте 25 микролитров раствора антител в отрицательные контрольные лунки в 384-луночном планшете. В пробирке объемом 15 миллилитров смешайте девять миллилитров раствора антител, 21,6 микролитра раствора FMLP и 1,7784 миллилитра нейтрофильной среды.Добавьте 25 микролитров этой смеси в положительные контрольные и тестовые лунки в 384-луночном планшете. С помощью многоканальной пипетки перелейте пять микролитров составного раствора с планшета библиотеки соединений на планшет с 384 лунками. Центрифугируйте суспензию при 500 г в течение одной минуты.Добавьте 20 микролитров суспензии нейтрофилов в каждую лунку с помощью 16-канальной пипетки. Инкубируйте пластину на шейкере при 300 об/мин в течение 10 минут. Затем, чтобы зафиксировать ячейки, добавьте в каждую лунку по три микролитра 16%-ного параформальдегида и инкубируйте на льду в течение 10 минут.

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.