CellTiter-Glo® 3D: A Sensitive, Accurate Viability Assay for
3D Cell Cultures
ABSTRACT
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 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 absorbance or fluorescence-based methods. In addition,
the assay protocol is simple and fast, giving results in as little as 30 minutes.
Michael P. Valley, Chad A. Zimprich, and Dan F. Lazar.
Promega Corporation
Publication Date: May 2014, tpub 142
Measuring Cell Viability in Microtissue Spheroids
Two-dimensional cell culture has long served as an invaluable tool for in vitro experiments. However, results derived from
studying flat monolayers of cells adhered to plastic often do not properly reflect the in vivo situation. Various characteristics of
actual tissue, such as morphology, cell-cell communication, gene expression, and biological responsiveness are much better
represented by microtissues produced under 3D cell culture conditions that replicate these biological functions. Much effort
has gone into the development of the variety of 3D cell culture models available, but little effort has gone into developing the
reagents to effectively assay these models. There is a significant risk to data quality in assuming that assays designed for 2D
monolayers will work equally well for 3D microtissues, especially considering the increased need for reagent penetration in
microtissue spheroids. The CellTiter-Glo® 3D Assay is a new, single-component, liquid reagent for measuring cell viability. The
assay has increased lytic capacity and is designed specifically for use with microtissues produced through 3D cell culture.
CellTiter-Glo® 3D Assay: Better Microtissue Penetration and Lytic Capacity
Initial studies with the classic CellTiter-Glo® Assay demonstrated effective lytic capacity with small microtissues. However, as
the microtissues became larger the classic reagent under-reported the total amount of ATP present—as judged by comparison
to results from a laborious trichloroacetic acid extraction procedure. Therefore, we optimized the CellTiter-Glo® Assay
formulation to increase lytic capacity and generate a new, 3D-optimized bioluminescent cell viability assay. As shown in Table
1, both the classic CellTiter-Glo® and the CellTiter-Glo® 3D Assay reagents recover a similar amount of ATP from small
HCT116 colon cancer spheroids (~200 µm diameter), but as the spheroids get larger (> 350 µm diameter), the CellTiter-Glo®
3D Reagent recovers 20–40% more ATP than the classic reagent. We have also observed similar increases in recovery for
other cell types, including HEK293 embryonic kidney cells and HepG2 liver carcinoma cells (data not shown).
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Valley, M., Zimprich, C.A., and Lazar, D.F.
Table 1. ATP Detected by the Classic CellTiter-Glo® and CellTiter-Glo® 3D Assays. Different amounts of HCT116 cells
(RPMI +10% FBS) were seeded in the InSphero GravityPLUS™ 96-well hanging-drop platform and grown for 4 days.
Microtissues 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.
ATP Content is Proportional to DNA Content and Viable Cell Number
As with the classic CellTiter-Glo® Reagent, the CellTiter-Glo® 3D Reagent generates a luminescent signal that is proportional
to the amount of ATP present using pure ATP (Figure 1, Panel A), and the amount of ATP is directly proportional to the number
of cells present in 2D cell culture (Figure 1, Panel B).
Figure 1. Luminescence generated by the CellTiter-Glo® Assay is proportional to ATP and/or cell number. Panel
A. Different concentrations of ATP in water were plated at 100 µl and assayed using the classic CellTiter-Glo® or
CellTiter-Glo® 3D Reagents. Panel B. Dilutions of Jurkat cells were prepared in RPMI 1640 with 10% FBS and plated at
100 μl cells per well. An equivalent volume of CellTiter-Glo® 3D Reagent was added to all samples, and after 2 minutes of
shaking, the luminescence was recorded at 10 minutes.
However, for microtissues in 3D culture, the correlation between cells seeded and luminescent signal is often not linear. For
example, with hanging-drop spheroids produced using the InSphero GravityPLUS™ 3D Culture and Assay Platform (Figure 2,
Panel A), effects such as contact inhibition on cell proliferation and reduced metabolic activity in the central region of large
spheroids cause the relationship between seeded cell number and luminescent signal to be curvilinear (Figure 2, Panel B).
However, by including CellTox™ Green Dye, a cell membrane-impermeant fluorogenic DNA-binding dye, in our lytic assay we
can show that as microtissue size increases, the amount of ATP detected (luminescence, RLU) by the CellTiter-Glo® 3D
Assay is proportional to the amount of DNA detected (fluorescence, RFU; Figure 2, Panel C). This confirms that the CellTiter-
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Valley, M., Zimprich, C.A., and Lazar, D.F.
Glo® 3D Assay is effectively lysing cells, resulting in release of ATP, and can accurately report the number of viable cells in
microtissues produced through 3D cell culture.
Figure 2. Seeding density vs. size, ATP content and DNA content. Panel A. Differential contrast images of HCT116 colon
cancer spheroids grown for 4 days in the InSphero GravityPLUS™ 96-well hanging-drop platform after seeding with 400, 800,
1600, 2400, 3200, 4800, 6400, or 8000 cells. Images were acquired in an InSphero GravityTRAP™ plate. Panels B and C. An
equivalent volume of CellTiter-Glo® 3D Reagent was added to all samples, and after 5 minutes of shaking, luminescence and/or
fluorescence was recorded at 30 minutes. In Panel C, a 2X concentration of CellTox™ Green Dye was added to the CellTiterGlo® 3D Reagent prior to sample addition.
CellTiter-Glo® 3D Assay Performance Relative to Other Viability Assays
The CellTiter-Glo® 3D Assay is significantly brighter than a competing bioluminescent ATP detection assay for measuring cell
viability (Figure 3, Panel A). As shown in Table 2, this difference is due to the recovery of considerably more ATP from both
small and large microtissues by the CellTiter-Glo® 3D Reagent. This is likely due (in part) to greater preservation of the
ATP because of the composition of the CellTiter-Glo® 3D Reagent, but also to a striking difference in lytic effectiveness
(Figure 3, Panel B). When CellTox™ Green Dye is added to each reagent prior to addition to the spheroid samples, confocal
laser fluorescent microscopy of ~300 µm HCT116 spheroids illustrates that the CellTiter-Glo® 3D Reagent penetrates almost
completely through the entire spheroid thickness. In contrast, the competitor reagent only penetrates the outer third of the
same-sized spheroids.
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Valley, M., Zimprich, C.A., and Lazar, D.F.
Figure 3. Lytic capacity of CellTiter-Glo® 3D compared to ATPlite 1Step Reagent. 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 each CellTiter-Glo® 3D Reagent (left) or ATPlite™
1Step Reagent (right) prior to sample addition, and after 5 minutes of shaking, the confocal laser fluorescent microscopy images
were acquired at 30 minutes using Ex/Em: 488/520nm. The spheroids in Panel B are ~300 µm in diameter, and the bars in each
image represent a distance of 200 µm.
Table 2. ATP Detected by the ATPlite™ 1Step and CellTiter-Glo® 3D Assays. Different amounts of HCT116 cells (RPMI
+10% FBS) were seeded in the InSphero GravityPLUS™ 96-well hanging-drop platform and grown for 4 days.
Microtissues were assayed by adding an equal volume of reagent to medium, shaking for 5 minutes and recording
luminescence after 30 minutes with a GloMax® luminometer. The amount of ATP detected was determined by
comparison with ATP standards.
Greater Sensitivity, Shorter Protocol
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Valley, M., Zimprich, C.A., and Lazar, D.F.
In common with many bioluminescent assays, the CellTiter-Glo® 3D Assay exhibits a signal that is orders of magnitude above
background (Figure 4). In contrast, 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. In addition to this
enhanced sensitivity, the CellTiter-Glo® 3D Assay has a much shorter assay time. The luminescent assay can be measured in
30 minutes or less, whereas alamarBlue® and MTT assays typically require multiple-hour incubations.
Figure 4. Performance of the CellTiter-Glo® 3D Assay compared to other viability assays. Signal-to-background ratios for
the luminescent CellTiter-Glo® 3D Assay compared to fluorescence (alamarBlue®) and absorbance (MTT) assays. Panel A.
HCT116 colon cancer cells (~340 µm) grown for 4 days in the InSphero GravityPLUS™ 96-well hanging-drop platform. Panel B.
InSphero human liver microtissues (~250 µm). All microtissues were assayed according to the assay manufacturers protocols.
Total assay times for the CellTiter-Glo® 3D, alamarBlue®, and MTT assays were 30 minutes, 3 hours, and 8 hours, respectively.
Summary
The CellTiter-Glo® 3D Assay is a new, validated bioluminescent assay for measuring the viability of cells produced through 3D
cell culture. It provides a fast, easy-to-use, single-component liquid reagent with superior lytic capacity for greater preservation
of ATP and enhanced penetration into 3D microtissues. This new reagent is more robust than our classic CellTiter-Glo® Assay
or competing ATP viability assays and offers superior sensitivity for 3D cell culture.
HOW TO CITE THIS ARTICLE
Scientific Style and Format, 7th edition, 2006
Valley, M., Zimprich, C.A., and Lazar, D.F. CellTiter-Glo® 3D: A Sensitive, Accurate Viability Assay for 3D Cell Cultures. [Internet] May
2014, tpub 142. [cited: year, month, date]. Available from: http://www.promega.com/resources/pubhub/a-cell-viability-assay-for-3dcultures/
American Medical Association, Manual of Style, 10th edition, 2007
Valley, M., Zimprich, C.A., and Lazar, D.F. CellTiter-Glo® 3D: A Sensitive, Accurate Viability Assay for 3D Cell Cultures. Promega
Corporation Web site. http://www.promega.com/resources/pubhub/a-cell-viability-assay-for-3d-cultures/ Updated May 2014, tpub 142.
Accessed Month Day, Year.
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Valley, M., Zimprich, C.A., and Lazar, D.F.
CellTiter-Glo and GloMax are registered trademarks of Promega Corporation. CellTox is a trademark of Promega Corporation.
alamarBlue is a registered trademark of Life Technologies Corporation. GravityPLUS is a trademark of InSphero AG.
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Valley, M., Zimprich, C.A., and Lazar, D.F.