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 av β2-integrinaktiveringshämmare hos humana neutrofiler

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 However, efalizumab was withdrawn due to its lethal side effect - progressive multifocal leukoencephalopathy resulting from JC virus reactivation6,7.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

Författare Spotlight: Utveckling av en metod för att identifiera små molekylära antagonister för β2-integrinaktivering 

En flödescytometribaserad high-throughput-teknik för screening av integrinhämmande läkemedel

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 med hög genomströmning av föreningar med hjälp av neutrofiler isolerade från humant blod i ett 384-brunnars plattformat

Använd en serologisk pipett och lägg försiktigt fyra milliliter hepariniserat humant blod över åtta milliliter densitetsgradientmedium i ett 15 milliliters centrifugrör. Centrifugera blodet vid 550 G i 30 till 50 minuter vid 20 grader Celsius. Använd sedan en enmilliliters pipett för att ta bort det övre lagret som innehåller plasma och mononukleära celler.Samla neutrofiler från det nedre grumliga bandet och cirka tre till fyra milliliter under den klara vätskan i ett 15 ml centrifugrör som innehåller 10 ml PBS. Vänd försiktigt upp och ner på slangen två till tre gånger för att blanda neutrofilsuspensionen. Centrifugera neutrofilsuspensionen vid 400 G i 10 minuter vid 20 grader Celsius, dekantera sedan försiktigt supernatanten från röret och återsuspendera pelleten i fem milliliter PBS.Centrifugera suspensionen vid 300 G i 10 minuter vid 20 grader Celsius. Efter att supernatanten har avlägsnats, använd vakuumsug för att eliminera kvarvarande supernatant på sondens vägg och runt sondens mynning. Återsuspendera pelleten i en milliliter neutrofilmedium.Kombinera en milliliter 1,2 mikrogram per milliliter antikroppslösning med 0,2 milliliter neutrofilmedium i ett 1,5 ml-rör. Tillsätt 25 mikroliter antikroppslösning till de negativa kontrollbrunnarna i en 384-hålsplatta. I ett 15 milliliters rör, kombinera nio milliliter av antikroppslösningen, 21,6 mikroliter av FMLP-lösningen och 1,7784 milliliter neutrofilmedium.Tillsätt 25 mikroliter av denna blandning till de positiva kontroll- och testbrunnarna i en 384-brunnsplatta. Använd en flerkanalspipett och överför fem mikroliter sammansatt lösning från den sammansatta biblioteksplattan till 384-brunnsplattan. Centrifugera suspensionen vid 500 G i en minut.Tillsätt 20 mikroliter neutrofilsuspension till varje brunn med hjälp av en 16-kanals pipett. Inkubera plattan på en shaker vid 300 varv per minut i 10 minuter. För att fixera cellerna, tillsätt sedan tre mikroliter 16% paraformaldehyd till varje brunn och inkubera på is i 10 minuter.

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.