A Flow Cytometry-Based High-Throughput Technique for Screening Integrin-Inhibitory Drugs

Summary

This protocol describes a flow cytometry-based, high-throughput screening method to identify small-molecule drugs that inhibit β2 integrin activation on human neutrophils.

Abstract

This protocol aims to establish a method for identifying small molecular antagonists of β2 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. β2 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 β2 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 β2 integrins, is utilized to quantify β2 integrin activation on isolated primary human neutrophils. N-formylmethionyl-leucyl-phenylalanine (fMLP) is used as a stimulus to activate neutrophil β2 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 β2 integrin inhibition are assessed within 3 h. Molecules that directly target β2 integrins or target molecules in the G protein-coupled receptor-initiated integrin inside-out activation signaling pathway can be identified through this approach.

Introduction

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 β2 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 diseases^3,

Protocol

Heparinized whole-blood samples were obtained from de-identified healthy human donors after obtaining informed consent, as approved by the Institutional Review Board of UConn Health, following the principles of the Declaration of Helsinki. Informed consent was obtained from all donors. The inclusion/exclusion criteria for this study were carefully developed to ensure the suitability of participants and to minimize potential risks. Eligible participants were aged between 18 and 65 years, of any ethnicity, fluent in Englis.......

Discussion

The initiation and termination of neutrophil stimulation and staining are determined by the addition of neutrophils and the fixative PFA. Therefore, ensuring the same time interval between pipetting neutrophils or PFA into each column is critical. This ensures that the stimulation and staining time of neutrophils from each well remains consistent. Due to the short lifespan of neutrophils, the entire experiment, from collecting blood from donors to completing flow cytometry, must be carried out on the same day. Neutrophil.......

Materials

Name	Company	Catalog Number	Comments
16-channel pipettes	Thermo	4661090N	Instrument
384-well plate	Greiner	784201	Materials
APC anti-human CD11a/CD18 (LFA-1) Antibody Clone: m24	BioLegend	363410	Reagents
Bravo Automated Liquid Handling Platform	Agilent	16050-102	384 multi-channel liquid handler
Centrifuge	Eppendorf	Model 5810R	Instrument
FlowJo	Becton, Dickinson & Company	NA	Software
Human Serum Albumin Solution (25%)	GeminiBio	800-120	Reagents
Lifitegrast	Thermofisher	50-208-2121	Reagents
Nexinhib20	Tocris	6089	Reagents
N-Formyl-Met-Leu-Phe (fMLP)	Sigma	F3506	Reagents
Paraformaldehyde 16% solution	Electron Microscopy Sciences	15710	Reagents
Plate buckets	Eppendorf	UL155	Accessory
Plate shaker	Fisher	88-861-023	Instrument
PolymorphPrep	PROGEN	1895 (previous 1114683)	Reagents
Prestwick Chemical Library Compound Plates (10 mM)	Prestwick Chemical Libraries	Ver19_384	1520 small molecules, 98% marketed approved drugs (FDA, EMA, JAN, and other agencies approved)
RPMI 1640 Medium, no phenol red	Gibco	11-835-030	Reagents
Swing-bucket rotor	Eppendorf	A-4-62	Rotor
ZE5 Cell Analyzer	Bio-Rad Laboratories	Model ZE5	Instrument