3D InSight™ Cytotoxicity Assay Kit

Monitor cell viability in 3D microtissues with Promega technology

The 3D InSight™ Cytotoxicity Assay Kit is a homogeneous biochemical assay for measuring total cellular ATP as an endpoint for cell viability.  The assay kit incorporates proprietary luminescent technology from Promega, and is validated for use with InSphero 3D InSight™ microtissues and culture media, providing a convenient 3D workflow to assess toxicity, drug efficacy, and more.

  • Rapid, homogeneous luminescent ATP assay for quantification of metabolically active cells
  • Proven lytic capacity compatible with 3D InSight™ microtissues and media including liver microtissues, pancreatic microislets and more
  • Provides convenience of ordering 3D microtissues, growth media and assay reagents from a single source

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    Catalog # Description
    AK-01-001-01 3D InSight™ Cytotoxicity Assay Kit
    Compatible Microtissues 
      3D InSight™ Human Liver Microtissues (96x)
      3D InSight™ Rat Liver Microtissues (96x)
      3D InSight™ HepG2 Liver Microtissues (96x)
      3D InSight™ Human Pancreatic Islet Microtissues (96x)
      3D InSight™ Tumor Microtissues
    Cell Culture Media 
     CS-07-001-xx 3D InSight™ Human Liver Maintenance Medium - TOX (for toxicity testing)
     CS-07-001a-xx 3D InSight™ Human Liver Maintenance Medium - AF (advanced formula for basic cultivation)
     CS-07-001b-xx 3D InSight™ Human Liver Maintenance Medium - INF (for inflammatory tox testing)
     CS-07-002-xx 3D InSight™ Rat Liver Maintenance Medium
     CS-07-005-xx 3D InSight™ Human Islets Maintenance Medium
     CS-07-006-xx 3D InSight™ Rat Islet Maintenance Medium
     CS-07-100-xx 3D InSight™ HepG2 Microtissue Maintenance Medium
     CS-07-101-xx 3D InSight™ Cell Line Maintenance Medium
  • Monitor spheroid viability over long-term culture

    Establishing novel 3D cell culture models requires characterization of microtissues to verify cell health and metabolic activity under varying culture conditions, including establishment and maintenance medium formulations, optimization of co-culture seeding densities, and spheroid longevity in culture.  The 3D InSight™ Cytotoxicity Assay Kit is a convenient assay for characterizing microtissue viability by measuring total spheroid ATP levels, an endpoint correlating to viable, metabolically active cells.  The graph below (left) displays a typical ATP-standard curve achieved using the kit components, while the figure below (right) displays ATP levels in 3D InSight™ Human Pancreatic Microislets after 6, 20, and 29 days of culture, displaying long-term viability of this 3D multi-cell type “pseudo-islet” model.




    Enable assessment of long-term & chronic toxicity

    The 3D InSight™ Cytotoxicity Assay Kit can be used to evaluate cytotoxicity in 3D microtissues, such as that seen following treatment of human liver microtissues with the drug Tolcapone. Tolcapone is known to be hepatotoxic following prolonged exposure in vivo. To simulate chronic exposure, 3D InSight™ Human Liver Microtissues received repeat dosing of various Tolcapone concentrations every 48 hours over 14 days, or for just 3 days of transient exposure. Total spheroid ATP levels were determined using 3D InSight™ Cytotoxicity Assay Kit at day 3 or 14, and normalized to vehicle control treated spheroids. Tolcapone was toxic with an IC50 of 12.45 μM following 14 day exposure, reflecting the chronic toxicity profile observed with the drug in vivo.

    Long term-tox Tolc d14


    Detect idiosyncratic toxicity in liver microtissue co-cultures

    The 3D InSight™ Cytotoxicity Assay Kit can be used to assess inflammation-associated toxicity, such as that seen in the benchmark compound Trovafloxacin. InSphero 3D InSight™ Liver Microtissue co-cultures contain both hepatocytes and non-parenchymal Kupffer cells, and are thus capable of simulating a pro-inflammatory environment following treatment with an inflammagen such as lipopolysaccharide (LPS). Total spheroid ATP content was assessed in individual Human Liver Microtissues using the 3D InSight™ Cytotoxicity Assay Kit, and normalized to vehicle control treated spheroids.  A clear shift in Trovafloxacin toxicity is revealed following treatment with LPS that is not seen without LPS (left), or following treatment with the non-toxic structurally-related compound Levofloxacin (with or without LPS, right).

    Idiosync TrovafloxacinIdiosync Levofloxacin

  •  AK-01-001-01 Kit

    Kit contents

    Each 3D InSight™ Cytotoxicity Assay Kit includes the following components, suitable for quantifying ATP in up to 4 x 96-well plates of microtissues, including controls:

    • CellTiter-Glo® 2.0 Reagent, 10 ml
    • 10 µM Adenosine-5’-triphosphate (ATP) standard, 750 µl
    • Product Manual with optimized protocol for 3D microtissues
    • Industry-leading support from our 3D cell culture experts


    Intended use

    The 3D InSight™ Cytotoxicity Assay Kit protocol is optimized for determination of ATP content in small microtissues, such as 3D InSight™ Liver Microtissues and 3D InSight™ Pancreatic Microislets.  The CellTiter-Glo® 2.0 reagent included in the kit offers optimal stability and luciferase activity, providing convenience for shipping, long term storage and assay planning.

    Note: InSphero recommends use of the Promega CellTiter-Glo® 3D Cell Viability Assay for determining ATP content in larger spheroids, such as those generated with actively proliferating tumor cell lines, as larger spheroids may exceed the lytic capacity of the assay reagent provided in the 3D InSight™ Cytotoxicity Assay Kit.

    Assay workflow


    Stability and storage

    All reagents are stable at +4°C through the expiration date listed on the assay kit (~ 4 weeks).  DO NOT FREEZE reagents and standards, or store them at room temperature, as this may result in loss of luciferase activity.

  • Support

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    Documents and downloads

    Product Manual

    Product Brochure

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