CellTiter-Glo® 3D Cell Viability Assay

How Does CellTiter-Glo 3D Work?

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. The simple, 30-minute protocol and ready-to-use reagent allows for fast results.

Learn how the CellTiter-Glo® 3D Cell Viability Assay can be used to assess drug toxicity in organoids.

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Better ATP Recovery from Larger 3D Microtissue Spheroids

Diameter of Spheroid (μm)	Classic CellTiter-Glo® Assay (pmol/microtissue)	CellTiter-Glo® 3D Assay (pmol/microtissue)	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

Better Lytic Capacity

Improved 3D Microtissue Penetration, More Accurate Viability Data

HCT116 colon cancer spheroids were generated by seeding cells in the InSphero GravityPLUS™ 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.

CellTiter-Glo 3D Culture & Better Lytic Capacity

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.

Sensitivity & CellTiter-Glo 3D Cell Culture — InSphero Insight™ human liver microtissues (~250μm). All microtissues were assayed using the CellTiter-Glo® 3D, alamarBlue® and MTT assays according to manufacturer's protocols. Total assay times for the CellTiter-Glo® 3D, alamarBlue® and MTT assays were 30 minutes, 3 hours and 8 hours, respectively.

Sensitivity & CellTiter 3D Spheroid Assay Reagent — HCT116 colon cancer cells seeded into an InSphero GravityPLUS™ 96-well hanging-drop platform and grown to generate ~340μm spheroids.

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.

3D Cell Culture Spheroid Assay Hanging Drop Method

3D Cell Culture Spheroid Assay Reagent Matrigel

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, and then assayed with the CellTiter-Glo® 3D Reagent. Luminescence was recorded at 30 minutes.

The CellTiter-Glo® 3D Assay Demonstrates Excellent Precision

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.

CellTiter-Glo 3D Culture & Spheroid Assay Precision

Tools to Monitor Biology in 3D Culture

When working with 3D culture models, choosing the right assay system is crucial. Learn about tools to monitor biology in 3D culture.

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Protocols

Complete Protocol

CellTiter-Glo® 3D Cell Viability Assay Technical ManualPDF (1749 KB) – English

Quick Protocols

FB258 CellTiter-Glo 3D Cell Viability AssayPDF (556 KB) – English

What's in the box

Item	Part #	Size
CellTiter-Glo® 3D Reagent	G968A	1 × 10ml

SDS

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Certificate of Analysis

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Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

Patents and Disclaimers

U.S. Pat. Nos. 7,741,067 and 8,361,739.

What's in the box

Item	Part #	Size
CellTiter-Glo® 3D Reagent	G968A	10 × 10ml

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

Patents and Disclaimers

U.S. Pat. Nos. 7,741,067 and 8,361,739.

What's in the box

Item	Part #	Size
CellTiter-Glo® 3D Reagent	G968B	1 × 100ml

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

Patents and Disclaimers

U.S. Pat. Nos. 7,741,067 and 8,361,739.

Ressources

Articles

Citations

Effect of Spheroidal Age on Sorafenib Diffusivity and Toxicity in a 3D HepG2 Spheroid Model.

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2019 Sci. Rep.
See all citations

Other

Frequently Used With

ROS-Glo™ H2O2 Assay

A homogeneous, fast and sensitive bioluminescent assay that measures the level of hydrogen peroxide directly in cell culture or in defined enzyme reactions.

G8820, G8821

GloMax® Discover System

High-performance microplate reader for detecting luminescence, fluorescence and absorbance.

GM3000

CellTiter-Glo® 3D Cell Viability Assay

A Cell Viability Assay Validated for 3D Microtissue Cultures

How Does CellTiter-Glo 3D Work?

Better ATP Recovery from Larger 3D Microtissue Spheroids

Better Lytic Capacity

Improved 3D Microtissue Penetration, More Accurate Viability Data

More Sensitive than Colorimetric or Fluorometric Cell Viability Assays

Compatible with a Variety of 3D Culture Methods

The CellTiter-Glo® 3D Assay Demonstrates Excellent Precision

Z'-factor Experiment With 3D Microtissues

Tools to Monitor Biology in 3D Culture

Bioluminescence Resource Center

Protocols

Complete Protocol

Quick Protocols

Specifications

What's in the box

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Patents and Disclaimers

Specifications

What's in the box

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Patents and Disclaimers

Specifications

What's in the box

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Patents and Disclaimers

Ressources

Articles

Citations

Other

CellTiter-Glo® 3D Cell Viability Assay

A Cell Viability Assay Validated for 3D Microtissue Cultures

How Does CellTiter-Glo 3D Work?

Better ATP Recovery from Larger 3D Microtissue Spheroids

Better Lytic Capacity

Improved 3D Microtissue Penetration, More Accurate Viability Data

More Sensitive than Colorimetric or Fluorometric Cell Viability Assays

Compatible with a Variety of 3D Culture Methods

The CellTiter-Glo® 3D Assay Demonstrates Excellent Precision

Z'-factor Experiment With 3D Microtissues

Tools to Monitor Biology in 3D Culture

Bioluminescence Resource Center

Protocols

Complete Protocol

Quick Protocols

Specifications

What's in the box

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Patents and Disclaimers

Specifications

What's in the box

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Patents and Disclaimers

Specifications

What's in the box

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Patents and Disclaimers

Ressources

Articles

Citations

Other

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Frequently Used With

ROS-Glo™ H2O2 Assay

GloMax® Discover System

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