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  • Who we serve
    • Academia
      Explore BioLegend

      Learn about our world class antibodies for a diverse set of research areas including immunology, neuroscience, cancer, stem cells and cell biology.

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    • Pharma / Biotech
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  • Products
    • Research & Development
      • Genomic Analysis
        • IncRNA in Functional Studies
        • Nucleic Acid Isolation
        • NGS Workflows
        • Microplate Readers
        • Microfluidic Nucleic Acid Analysis
        • CRISPR Technologies
        • RNA Interference
        • Transfection and Ancillary Reagents
        • Oligonucleotide Custom Synthesis
        • cDNAs and ORF Clones
        • Single-Cell Sequencing
        • Labeled Nucleotides
        • qPCR
        • Library Prep Kits
        • Magnetic Beads
        • Viral Vector Products
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      • Protein Analysis
        • Binding Assays
        • Immunoassays
        • Microfluidic Protein Characterization
        • Microplate Readers
        • Microplates
        • Recombinant Proteins
        • Western Blotting
        • Sample Dissociation
        • M-PVA Magnetic Beads
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      • Cell Analysis
        • Cell Isolation
        • Cell Lines & Stem Cells
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        • Cell Health & Viability
        • Cellular Imaging & Analysis
        • Immunoassays
        • In Vivo Imaging
        • Microplates
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      • Research Solutions
        • Agrigenomics Workflows
        • Assay Development Workflows
        • Biobanking Workflows
        • Biologics
        • Biomarker Discovery
        • Cell and Gene Therapy
        • Disease Research
        • Drug Development
        • Functional Genomic Screening Solutions
        • GMP Workflows
        • Immunoassays Solutions
        • Microphysiological Systems
        • pHSense Reagents
        • Precision Medicine Research
        • Small Molecule Drug Discovery
        • Target Class
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    • Clinical & Diagnostics
      • Reproductive Health
        • Maternal and Prenatal Testing
        • Revvity Genomics Software
        • Newborn Screening
        • Newborn Sequencing Research
        • Pregnancy-Relevant Infections
        • Endocrine Reproductive
        • Cell-free DNA Analysis
        • Neonatal Research
        • Preimplantation Genetic Testing
        • PlGF Testing Research
        • Molecular Cytogenetics
        New IVD Mimix™ reference standards for oncology workflows.

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      • Infectious Diseases
        • Tuberculosis Management
        • EUROIMMUN Solutions
        • IDS Solutions
        • Nucleic Acid Isolation for Pathogen Detection
        • Cytomegalovirus
        • SARS-COV-2 Testing Solutions
        • Bacterial & Viral Nucleic Acid Isolation
        • Metagenomics
        New IVD Mimix™ reference standards for oncology workflows.

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      • Cancer
        • ASR Flow Cytometry Antibodies
        • HPV Testing
        • ctDNA Workflows
        • miRNA-seq Analysis
        • Exosome/cfRNA Analysis
        • Targeted Sequencing
        • Mimix Reference Standards
        • Functional Testing
        NEW! Mimix IVD reference standards

        Providing diagnostic labs with trusted quality controls for developing tests and monitoring workflows, with the added assurance of IVD marking. Explore our newest MimixTM controls.

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      • Autoimmunity
        • EUROIMMUN Solutions
        • IDS Solutions
        New IVD Mimix™ reference standards for oncology workflows.

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      • Endocrinology
        New IVD Mimix™ reference standards for oncology workflows.

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      • Allergy
        New IVD Mimix™ reference standards for oncology workflows.

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      • Neurodegeneration
        • Alzheimer's Disease
        • Amytrophic Lateral Sclerosis (ALS)
        • Neurogranin
        • BACE1
        New IVD Mimix™ reference standards for oncology workflows.

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      • Rapid Patient Testing
        New IVD Mimix™ reference standards for oncology workflows.

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      New IVD Mimix™ reference standards for oncology workflows.

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    • Reagents
      • BioLegend
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      • Cellular Imaging Reagents
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      • chemagic Nucleic Acid Purification Kits
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      • CRISPR Technologies
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      • GPCR Research Reagents
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      • Immunoassays
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      • In Vivo Imaging Reagents
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      • Liquid Scintillation Cocktails
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      • NGS Library Prep Kits
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      • pHSense Reagents
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      • qPCR
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      • Radiochemicals
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      • RNA Interference
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      • Primary and Secondary Antibodies
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    • Platforms & Automation
      • Nucleic Acid Purification
        • chemagic Applications
        • chemagic IVD Instruments
        • chemagic IVD Kits
        • chemagic Instruments
        • chemagic Kits
        • M-PVA Magnetic Beads
        New! Cellometer™ Ascend™

        Explore our cell counting and image cytometry solutions.

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      • Automated Liquid Handling
        • Flow Cytometry Cocktail Prep Automation
        • Liquid Handling Workstations
        • Automated Liquid Handling Applications
        • Liquid Handling Consumables & Accessories
        New! Cellometer™ Ascend™

        Explore our cell counting and image cytometry solutions.

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      • Integrated Lab Automation
        • Automated Plate Loading
        • Workstation Modules
        • Scheduling & Control Software
        • Robotics
        New! Cellometer™ Ascend™

        Explore our cell counting and image cytometry solutions.

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      • Microfluidic Analysis
        • Microfluidic Protein Characterization
        • Microfluidic Nucleic Acid Analysis
        New! Cellometer™ Ascend™

        Explore our cell counting and image cytometry solutions.

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      • Detection Solutions
        • Plate Reader Accessories
        • Microplate Readers
        • Radiometric Detectors
        • Radiochemicals
        • Liquid Scintillation Cocktails
        • Radiometric Consumables & Accessories
        New! Cellometer™ Ascend™

        Explore our cell counting and image cytometry solutions.

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      • Imaging
        • Cellular Imaging & Analysis
        • In Vivo Imaging
        New! Cellometer™ Ascend™

        Explore our cell counting and image cytometry solutions.

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      • Cell Counting and Image Cytometry
        New! Cellometer™ Ascend™

        Explore our cell counting and image cytometry solutions.

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      • Sample Homogenization
        • Homogenizers
        • Homogenizer Accessories
        • Homogenizer Consumables
        • Sample Centrifugation
        • Sample Sonication
        New! Cellometer™ Ascend™

        Explore our cell counting and image cytometry solutions.

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      • In Vitro Diagnostics (IVD) Platforms & Automation
        • IVD Nucleic Acid Isolation
        • IVD Automated Liquid Handling
        • IVD Radiometric Detectors
        • EUROIMMUN Instruments
        • IDS Instruments
        • Clinical Applications
        New! Cellometer™ Ascend™

        Explore our cell counting and image cytometry solutions.

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      • Consumables & Accessories
        • Cassettes
        • Cell Harvesters
        • In Vivo Imaging Accessories
        • High-Content Imaging Accessories
        • Microplates
        • Radioactive Spill Cleaners
        • Scintillation Cocktails
        • Transfer Membranes
        • Charcoal Traps
        • Homogenizer Accessories
        • Homogenizer Consumables
        New! Cellometer™ Ascend™

        Explore our cell counting and image cytometry solutions.

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      • Instrument Service & Maintenance
        • On Demand Equipment Service
        • Equipment Service Plans
        New! Cellometer™ Ascend™

        Explore our cell counting and image cytometry solutions.

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      New! Cellometer™ Ascend™

      Explore our cell counting and image cytometry solutions.

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    • Consumables & Accessories
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    • Signals Software
      • All Products
        • Signals One
        • ChemDraw
        • Spotfire®
        • Signals Clinical
        • Signals Notebook
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      • All Solutions
        • Biology
        • Discovery Chemistry
        • Clinical & Translational
        • Specialty Chemicals
        • Food/Flavor/Fragrance
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    • Revvity Omics Services
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  • Services
    • Preclinical Services
      • Antibody Drug Conjugate Services
        Preclinical services

        Work with our experienced scientific team and leverage our advanced technologies to help accelerate the preclinical drug discovery process.

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      • Complex Cell Model Screening Services
        Preclinical services

        Work with our experienced scientific team and leverage our advanced technologies to help accelerate the preclinical drug discovery process.

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      • Base Editing Platform
        • Pin-point base editing platform cell line engineering services
        • Pin-point base editing platform pooled tiled screening services
        Preclinical services

        Work with our experienced scientific team and leverage our advanced technologies to help accelerate the preclinical drug discovery process.

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      • Immune Cell Screening
        Preclinical services

        Work with our experienced scientific team and leverage our advanced technologies to help accelerate the preclinical drug discovery process.

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      • Functional Genomic Screening Services
        Preclinical services

        Work with our experienced scientific team and leverage our advanced technologies to help accelerate the preclinical drug discovery process.

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      • Cell Panel Screening
        Preclinical services

        Work with our experienced scientific team and leverage our advanced technologies to help accelerate the preclinical drug discovery process.

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      • Cell Line Engineering
        • CHOSOURCE CLE Services
        Preclinical services

        Work with our experienced scientific team and leverage our advanced technologies to help accelerate the preclinical drug discovery process.

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      • Viral Vector Engineering and Manufacture
        Preclinical services

        Work with our experienced scientific team and leverage our advanced technologies to help accelerate the preclinical drug discovery process.

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      Preclinical services

      Work with our experienced scientific team and leverage our advanced technologies to help accelerate the preclinical drug discovery process.

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    • Revvity Omics Services
      • Revvity Omics Clinical Services
        • Cytogenomics
        • Global Laboratory Network
        • Metabolic Testing
        • Newborn Screening Services
        • Prenatal Screening Services
        • Proactive Testing
        • Rare Disease Testing
        • Specialized and Customized Assays
        • Sponsored Testing Programs
        T-SPOT.TB testing services.

        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      • Revvity Omics Pharma Services
        T-SPOT.TB testing services.

        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      T-SPOT.TB testing services.

      Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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    • Clinical & Testing Services
      • Revvity Omics Clinical Services
        T-SPOT.TB testing services.

        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      • Revvity Omics Pharma Services
        T-SPOT.TB testing services.

        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      • Cellular and Humoral Immunoassays
        T-SPOT.TB testing services.

        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      • Tuberculosis Testing Services
        • Oxford Diagnostic Laboratories Service Closures
        T-SPOT.TB testing services.

        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      T-SPOT.TB testing services.

      Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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    • Customization Services
      • Assays and Reagents
        • Assay Development
        • Compound profiling
        T-SPOT.TB testing services.

        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      • Microplate Services
        T-SPOT.TB testing services.

        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      • Custom Conjugation & Labeling
        • Custom Labeling
        • Small Molecule
        T-SPOT.TB testing services.

        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      • Radiosynthesis and Labeling
        T-SPOT.TB testing services.

        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      T-SPOT.TB testing services.

      Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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    • Licensing
      • Gene Delivery Licensing
        CHOSOURCE expression platform

        Revvity's expression platform provides an enhanced system for the development and manufacturing of biotherapeutics that can be used in commercial manufacturing applications.

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      • Gene Expression Systems
        • AAV Production Platform
        • CHOSOURCE Platform
        CHOSOURCE expression platform

        Revvity's expression platform provides an enhanced system for the development and manufacturing of biotherapeutics that can be used in commercial manufacturing applications.

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      • Pin-point™ Base Editing Platform
        CHOSOURCE expression platform

        Revvity's expression platform provides an enhanced system for the development and manufacturing of biotherapeutics that can be used in commercial manufacturing applications.

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      • Virus Screening
        CHOSOURCE expression platform

        Revvity's expression platform provides an enhanced system for the development and manufacturing of biotherapeutics that can be used in commercial manufacturing applications.

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      CHOSOURCE expression platform

      Revvity's expression platform provides an enhanced system for the development and manufacturing of biotherapeutics that can be used in commercial manufacturing applications.

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    • Viral Vector Engineering and Manufacture
      • AAV Services
        T-SPOT.TB testing services.

        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      • Lentivirus Services
        T-SPOT.TB testing services.

        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      T-SPOT.TB testing services.

      Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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    • Instrument Service & Maintenance
      • AV Services
        T-SPOT.TB testing services.

        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      • Equipment Service Plans
        T-SPOT.TB testing services.

        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      • On-demand Equipment Service
        T-SPOT.TB testing services.

        Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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      T-SPOT.TB testing services.

      Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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    • Customer Training
      T-SPOT.TB testing services.

      Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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    • OEM Solutions
      T-SPOT.TB testing services.

      Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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    T-SPOT.TB testing services.

    Revvity's Oxford Diagnostic Laboratories is a large referral laboratory for tuberculosis testing services based on our T-SPOT technology.

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  • Company
    • Purpose
      • Approach
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      • Our Story
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    • Careers
      • Careers Home
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      • Why Revvity
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    • Investor Relations
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        Investor Day 2024

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        Investor Day 2024

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      • Stock Info
        Investor Day 2024

        Driving meaningful innovation that profoundly impacts science and human lives.

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      Investor Day 2024

      Driving meaningful innovation that profoundly impacts science and human lives.

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    • ESG
      • Environmental
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    • News
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  • Resources
    • Product Support
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  • Brands
      Revvity Brands
    • BioLegend

    • EUROIMMUN

    • Revvity Omics

    • Revvity Signals

    • Tulip Diagnostics

    • ViaCord

    • Popular Revvity Product Brands
    • AlphaLISA and SureFire Ultra

    • BioQule NGS

    • chemagic

    • EnVision Nexus

    • Fontus

    • HTRF

    • IVIS

    • LEGENDplex

    • Opera Phenix

    • T-SPOT

    • TotalSeq

    • View all Revvity brands
  • Welcome to Revvity: renowned brands and boundless innovation.

    Hearing the word "can't" is our call to action!

    We help scientists, researchers, and clinicians overcome the world's greatest health obstacles.

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    Featured brand: BioLegend

    Learn about our world-class antibodies for a diverse set of research areas including immunology, neuroscience, cancer, stem cells and cell biology.

    Visit BioLegend.com

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  • HTRF Human and Mouse Total ATG16L1 Detection Kit, 10,000 Assay Points

HTRF Human and Mouse Total ATG16L1 Detection Kit, 10,000 Assay Points

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HTRF Human and Mouse Total ATG16L1 Detection Kit, 10,000 Assay Points
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Photo ATG16L1 Total Kit
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This HTRF kit enables the cell-based quantitative detection of total ATG16L1 as a readout of the autophagy pathway, and can be combined with our HTRF Phospho-ATG16L1 (Ser278) kit.

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Feature Specification
Application Cell Signaling
Sample Volume 16 µL
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This HTRF kit enables the cell-based quantitative detection of total ATG16L1 as a readout of the autophagy pathway, and can be combined with our HTRF Phospho-ATG16L1 (Ser278) kit.

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Product variants
Unit Size: 500 assay points
Part #:
64ATG16TPEG
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USD 2,237.17
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Unit Size: 10,000 assay points
Part #:
64ATG16TPEH
List price
USD 13,214.42
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For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).
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Photo ATG16L1 Total Kit
Photo ATG16L1 Total Kit
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HTRF Human and Mouse Total ATG16L1 Detection Kit, 10,000 Assay Points
Photo ATG16L1 Total Kit
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Photo ATG16L1 Total Kit
Photo ATG16L1 Total Kit
HTRF Human and Mouse Total ATG16L1 Detection Kit, 10,000 Assay Points
Photo ATG16L1 Total Kit
Photo ATG16L1 Total Kit
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Product information

  • Overview
  • How it works
  • Assay validation
  • Simplified pathway
  • Specifications
  • Video gallery

Overview

Autophagy Related 16 Like 1 (ATG16L1) is a protein that is essential for autophagy. ATG16L1 is recruited to the phagophore and recruits ATG5-ATG12 complex to a phagophore, facilitating LC3 lipidation (LC3-I to LC3-II conversion) and contributing to autophagosome maturation.

Phosphorylated ATG16L1 at Ser278 is only present on newly-forming autophagosomes, making its quantification an ideal surrogate for autophagic vesicle formation and autophagy induction. ATG16L1 is involved in conditions such as Alzheimer's Disease, colorectal cancer, heart disease, breast cancer, and chronic myeloid leukemia.

How it works

HTRF Total ATG16L1 assay principle

The HTRF Total ATG16L1 assay quantifies the expression level of ATG16L1 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based, and does not require gels, electrophoresis, or transfer. The HTRF Total ATG16L1 assay uses two labeled antibodies, one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of ATG16L1 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

1phospho-how-it-works-atg14-total-assay-principle.svg

 

HTRF Total ATG16L1 two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a low volume detection plate (either HTRF 384-lv or 96-lv plate) before the addition of HTRF Total ATG16L1 detection reagents. This protocol enables the cells' viability and confluence to be monitored

Two-plate protocol of the HTRF total BRD2 assay

 

HTRF Total ATG16L1 one-plate assay protocol

Detection of Total ATG16L1 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization, while maintaining robust HTRF quality.

One-plate protocol of the HTRF total BRD2 assay

 

Assay validation

Validation of HTRF total ATG16L1 and Phospho-ATG16L1 (Ser278) kits on the human U-87 MG cell line using mTOR and ULK1/2 inhibitors

Human U-87 MG cells were plated in a 96-well plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with a dose-response of AZD2014, an mTOR inhibitor, for 3 h at 37°C, 5% CO2. Following culture medium removal, cells were lysed with 25 µl of supplemented lysis buffer #3 (1X) + BR (2X) for 1 h at RT under gentle shaking. After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate, then 4 µL of the HTRF Phospho-ATG16L1 (Ser 278) or Total ATG16L1 detection reagents were added. The HTRF signals for the two kits were recorded after an overnight incubation at room temperature. As expected, AZD2014, a potent mTOR inhibitor, induced autophagy activation, leading to a dose-dependent increase in ATG16L1 phosphorylation on Serine 278 without any significant effect on the expression level of the total ATG16L1 protein. Validation of HTRF Total ATG16L1 and Phospho-ATG16L1 (Ser278) kits on the human U-87 MG cell line using mTOR and ULK1/2 inhibitors Human U-87 MG cells were plated in a 96-well plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with 2.5 µM of AZD2014, an mTOR inhibitor, and a dose-response of ULK-101, an autophagy kinase ULK1/2 inhibitor, for 3 h at 37°C, 5% CO2. Following culture medium removal, cells were lysed with 25 µl of supplemented lysis buffer #3 (1X) + BR (2X) for 1 h at RT under gentle shaking. After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate, then 4 µL of the HTRF Phospho-ATG16L1 (Ser 278) or Total ATG16L1 detection reagents were added. The HTRF signals for the two kits were recorded after an overnight incubation at room temperature. As expected, ULK-101, a potent and dual autophagy kinase ULK1/2 inhibitor, repressed autophagy activation induced by AZD2014, leading to a dose-dependent decrease in ATG16L1 phosphorylation on Serine 278, without any significant effect on the expression level of the ATG16L1 total protein.

2assay-validation-phospho-ser278-atg16l1-2-64atg16s8peg-64atg16s8peh-64atg16s8pef.svg
3assay-validation-phospho-ser278-atg16l1-3-64atg16s8peg-64atg16s8peh-64atg16s8pef.svg

 

Specificity of HTRF Total ATG16 assay using HAP1 ATG16 knock-out cells

The human total ATG16L1 expression level was assessed using the HTRF Total kit in Wild Type (WT) and ATG16 knock-out (KO) HAP1 cells. The cell lines were cultured in flasks and incubated 48 h at 37°C, 5% CO2. After culture medium removal, cells were lysed with supplemented lysis buffer #3 (1X) + BR (2X) for 30 min at RT under gentle shaking. After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate then 4 µL of the HTRF Total ATG16L1 detection reagents were added. In parallel, GAPDH level was monitor using the GAPDH Housekeeping Cellular Kit (# 64GAPDHPET/G/H). 4 µL of cell lysate were prediluted with 180 µL of diluent #11. Then, 16 µL of this diluted material were mixed with 4 µL of the HTRF GAPDH Housekeeping Cellular detection reagents. HTRF signal for both kits was recorded after an overnight incubation at room temperature. The measured level of GAPDH was equivalent under both tested conditions. In contrast, while the HTRF Total ATG16L1 signal was significantly positive in HAP1 WT cells, it was dramatically decreased (equivalent to the negative signal) in HAP1 ATG16 KO cells, demonstrating the specificity of the HTRF Total ATG16L1 assay for the detection of the ATG16L1 protein. HAP1 ATG16 KO (10bp deletion) cell line from Horizon Discovery # HZGHC000296c010 HAP1 WT cell line from Horizon Discovery # C631

4assay-validation-phospho-ser278-atg16l1-specificity-64atg16s8peg-64atg16s8peh-64atg16s8pef.svg

 

Versatility of HTRF Total ATG16L1 assay in cell lines from multiple species

Human (PC-3), mouse (C2C12) and rat (H4-II-E) cell lines were seeded at 100,000 cells/well in a 96-well plate. After a 24h incubation, the cells were lysed with 25 µL of supplemented lysis buffer #3 (1X) + BR (2X) for 1 h at RT under gentle shaking. After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate then 4 µL of the HTRF Total ATG16L1 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature. The HTRF Total ATG16L1 assay efficiently displayed significant positive signals in the cell lines from the 3 species tested, demonstrating its capability to detect human, mouse and rat versions of the protein, and its suitability for translational research.

1assay-validation-total-atg16l1-cross-reactivity-64atg16tpeg-64atg16tpeh-64atg16tpey.svg

 

HTRF Total ATG16L1 Detection Kit compared to Western Blot

HCT116 cells were cultured in flasks and incubated 48 h at 37°C, 5% CO2. Then, the cells were treated with 100 nM of Calyculin A for 10 min. After culture medium removal, cells were lysed with supplemented lysis buffer #3 (1X) + BR (2X) for 30 min at RT under gentle shaking. Soluble supernatants were collected after 10 min centrifuging. Equal amounts of lysates were used for a side-by-side comparison of WB and HTRF. The HTRF Total ATG16L1 assay is 4 times more sensitive than Western Blot assay.

6assay-validation-phospho-ser278-atg16l1-wb-comparison-64atg16s8peg-64atg16s8peh-64atg16s8pef.svg

 

Simplified pathway

Simplified ATG16 signaling pathway in macroautophagy

Autophagy is an evolutionary conserved cellular process that occurs in virtually all eukaryotic cells, ranging from yeast to mammals. Autophagy is crucial in the maintenance of homeostasis, being involved in numerous physiological processes including stress responses (e.g. starvation, hypoxia, high temperature), cell growth, and aging. Conversely, dysfunctions in autophagic mechanisms have been associated with diseases such as cancer, neurodegenerative diseases, infectious diseases, and cardiac and metabolic diseases. There are 3 types of autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy. Macroautophagy (hereafter referred to as autophagy) starts with the formation of a bud from lipid phosphatidylinositol 3-phosphate rich membranes such as reticulum endoplasmic, Golgi, or plasma membranes, and known as a pre-autophagosomal structure (PAS). This cup-shaped structure requires the ULK1/Atg1 complex formed by Atg13, Atg101, FIP200, and ULK1 proteins. A transmembrane protein, Atg9, contained in vesicles is involved in the recruitment of Atg2-Atg18 to the PAS. With its phospholipid transfer activity, the Atg2 protein supplies phospholipids necessary for membrane elongation. ULK1 phosphorylates and activates Beclin1, which partners with Vsp15, Vsp34, Atg14, and NRFB2/Atg38 to form a class III Phosphatidylinositol 3-kinase (PI3KC3-Complex 1). This complex leads to the production of phospho-inositol triphosphate (PIP3), which further directs the recruitment of PI3P binding Atg18/WIPI 1–4 proteins and the Atg12-Atg5-Atg16 complex. The latter complex, along with Atg4 and Atg7, is implicated in the LC3B lipidation (LC3-I to LC3-II conversion) on the phagophore. This contributes to autophagosome maturation, which subsequently fuses with lysosomes forming an autolysosome, where trapped cellular components are degraded and building blocks recycled. Phosphorylated ATG16L1 at Ser278 is only present on newly-forming autophagosomes, making its quantification an ideal surrogate for autophagic vesicle formation and autophagy induction. Importantly, its levels are not affected by prolonged stress or late-stage autophagy blocks, which can jeopardize autophagy analysis. Moreover, ATG16L1 phosphorylation by the autophagy kinase ULK1 is a conserved indicator of autophagy induction, which is activated by multiple stimuli. Taken together, analysis of ATG16L1 phosphorylation represents an excellent readout when studying autophagy induction.

7phospho-pathway-atg16l1-64atg16s8peg-64atg16s8peh-64atg16s8pef.svg

 

Specifications

Application
Cell Signaling
Brand
HTRF
Detection Modality
HTRF
Lysis Buffer Compatibility
Lysis Buffer 1
Lysis Buffer 3
Lysis Buffer 4
Molecular Modification
Total
Product Group
Kit
Sample Volume
16 µL
Shipping Conditions
Shipped in Dry Ice
Target Class
Phosphoproteins
Target Species
Human
Mouse
Technology
TR-FRET
Unit Size
10,000 assay points

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HTRF Human and Mouse Total ATG16L1 Detection Kit, 10,000 Assay Points
HTRF Human and Mouse Total ATG16L1 Detection Kit, 10,000 Assay Points

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