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Promega Corporation

CellTiter-Glo® 3D: A Cell Viability Assay Validated for 3D Microtissue Cultures

Accurate 3D Cytotoxicity Determination

Easy Assay Implementation

Simple, 30-Minute Protocol

The CellTiter-Glo® 3D Cell Viability Assay is designed for determining cell viability in 3D microtissue spheroids. The assay reagent penetrates large spheroids and has increased lytic capacity—allowing more accurate determination of viability compared to other assay methods.

Based on the same reliable chemistry as the classic CellTiter-Glo® Assay, this new 3D assay reagent measures ATP as an indicator of viability and generates a luminescent readout that is much more sensitive than colorimetric or fluorescence-based methods.

Improved 3D Microtissue Penetration, More Accurate Viability Data

The CellTiter-Glo® 3D Assay Reagent penetrates deeper into 3D microtissue spheroids, releases more ATP, and gives a more accurate measure of viability than kits designed for 2D cell culture.

Lytic capacity of CellTiter-Glo 3D compared to ATPlite 1Step Reagent 12330TB

Superior lytic capacity of CellTiter-Glo® 3D Reagent compared to ATPlite 1Step Reagent. HCT116 colon cancer spheroids were generated by seeding cells in the InSphero GravityPLUSTM 96-well hanging-drop platform and grown for 4 days. Panel A. An equivalent volume of reagent was added to all samples, and after 5 minutes of shaking, luminescence was recorded at 30 minutes. Panel B. A 2X concentration of CellTox™ Green Dye was added to CellTiter-Glo® 3D Reagent (left) or ATPlite™ 1Step Reagent (right) prior to sample addition as an indicator of cell lysis and images were acquired at 30 minutes. The spheroids in Panel B are ~300μm in diameter, and the bars in each image represent a distance of 200μm.

Better ATP Recovery from Larger 3D Microtissue Spheroids

Diameter of Spheroid (μm) ATP Recovered (pmol/microtissue)
Classic CellTiter-Glo® Assay CellTiter-Glo® 3D Assay Ratio
188 16 ± 4 17 ± 4 1.10
386 79 ± 3 94 ± 11 1.19
459 103 ± 2 126 ± 11 1.22
565 127 ± 3 178 ± 17 1.40

ATP detected by the classic CellTiter-Glo® and CellTiter-Glo® 3D Assays. Various numbers of HCT116 cells (RPMI +10% FBS) were seeded in the InSphero GravityPLUSTM 96-well hanging-drop platform and grown for 4 days to form a range of spheroid sizes. Microtissue spheroids were assayed by adding an equal volume of reagent to media volume, shaking for 5 minutes, and recording luminescence after 30 minutes using a GloMax® luminometer. The amount of ATP detected was determined by comparison with ATP standards.

CellTiter-Glo® 3D Assay is More Sensitive than Colorimetric or Fluorometric Cell Viability Assays

Luminescent signals from the CellTiter-Glo® 3D Assay are orders of magnitude above background. Other non-lytic viability assays that measure changes in fluorescence (e.g. alamarBlue®) or absorbance (e.g., MTT) generate signals that are only modestly higher than their no-cell control signals.

CellTiter-Glo 3D Assay compared to alamarBlue and MTT assays 12331MC

CellTiter-Glo® 3D Assay signal-to-background ratio is significantly better than colorimetric or fluorometric assays. Panel A. HCT116 colon cancer cells seeded into an InSphero GravityPLUS™ 96-well hanging-drop platform and grown to generate ~340μm spheroids. Panel B. InSphero Insight™ human liver microtissues (~250μm). All microtissues were assayed using the CellTiter-Glo® 3D, alamarBlue® and MTT assays according to manufacturers protocols. Total assay times for the CellTiter-Glo® 3D, alamarBlue® and MTT assays were 30 minutes, 3 hours and 8 hours, respectively.

Simple, 30-Minute Protocol and Ready-to-Use Reagent

  • Thaw single reagent
  • Equilibrate to room temperature
  • Add to 3D microtissue cell cultures
Overview of the CellTiter-Glo® 3D Cell Viability Assay protocol.

CellTiter-Glo® 3D is Compatible with a Variety of 3D Culture Methods

The results of compound screening using the CellTiter-Glo® 3D Assay in hanging-drop, ultra-low attachment plate (ULA) and Matrigel® 3D cultures are shown below. Equivalent results were achieved for all three methods.


HCT116 colon cancer cells were seeded as follows: 400 cells in hanging-drop; 1,000 cells in ULA or Matrigel®. Microtissues were grown for 4 days, treated with compounds for 48 hours, then assayed with the CellTiter-Glo® 3D Reagent. Luminescence was recorded at 30 minutes.

CellTiter-Glo® 3D Assay is Easy to Use in High-Throughput Screening Assays

  • Homogeneous, “Add-Mix-Read” protocol
  • Scalable to 96, 384, or 1536-well plates
  • Stable, “Glow-type” luminescent signal (half-life >3 hours) allows batch mode or consecutive processing of multiple plates

Assay Precision

The CellTiter-Glo® 3D Assay demonstrates excellent precision by Z´-factor determination,a measure of assay precision commonly used to evaluate the suitability of an assay for screening applications. A Z´-factor greater than 0.5 is considered acceptable, and the quality of the assay increases as the Z´-factor approaches 1.


Z´-factor experiment with 3D microtissues. Four hundred HCT116 colon cancer cells were seeded into each of 60 wells of a 96-well InSphero GravityPLUS™ hanging-drop plate and incubated for 4 days to form 60 spheroids (~350μm in diameter). Half of the spheroids were treated with 100μM panobinostat (black squares), and the other half were treated with vehicle (1% DMSO, orange squares). After 48 hours, all samples were assayed with the CellTiter-Glo® 3D Reagent. The CellTiter-Glo® 3D Assay provided a Z´-factor of 0.81.

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