Description
What Is the CellTiter-Glo® Assay and How Does It Work?
The CellTiter-Glo® Luminescent Cell Viability Assay determines the number of viable cells in culture by quantifying adenosine triphosphate (ATP)—an indicator of metabolically active cells. It uses a bioluminescent luciferase reaction: viable cells release ATP upon lysis, which reacts with the CellTiter-Glo® Reagent to produce a luminescent signal proportional to cell number. The entire protocol requires just one reagent addition and produces results in 10 minutes.
Biochemical Principle: ATP Quantification via Luciferase
ATP is a universal marker of cell viability. Living cells maintain ATP levels through active metabolism, while dead or dying cells rapidly lose ATP. The CellTiter-Glo® Reagent lyses cells on contact, releasing intracellular ATP. The released ATP then drives a luciferase-catalyzed reaction with luciferin, generating bioluminescence that is measured by a compatible luminometer or plate reader.
The luminescence produced is a "glow-type" signal with a half-life generally exceeding 5 hours, depending on cell type and medium. This extended half-life eliminates the need for reagent injectors and allows batch or continuous plate processing—a significant practical advantage in high-throughput environments.
How Do You Perform a CellTiter-Glo® Assay? Key Steps
The assay follows a simple three-step workflow that minimizes hands-on time and potential for error:
Add CellTiter-Glo® Reagent directly to cells in culture medium (no washing or medium removal required).
Mix contents for 2 minutes on an orbital shaker to lyse cells.
Incubate for 10 minutes to stabilize the luminescent signal, then record luminescence.
The homogeneous, "add-mix-measure" format eliminates errors common to multi-step ATP measurement protocols, making CellTiter-Glo® one of the most reproducible viability assays available for multiwell plate formats.
CellTiter-Glo® Assay offers 10-cell sensitivity; up to 5 logs linear range. The signal was read on a MyGlo® Reagent Reader .
Notes:
Using 100μl of CellTiter-Glo® Reagent per assay in a 96-well format, Cat.# G7570 is sufficient to perform 100 assays; Cat.# G7571 and G7572, 1,000 assays; Cat.# G7573, 10,000 assays. Using 25μl of CellTiter-Glo® Reagent per assay in a 384-well format, Cat.# G7570 is sufficient to perform 400 assays; Cat.# G7571 and G7572, 4,000 assays; Cat.# G7573, 40,000 assays.
What Types of Cells and Culture Models Are Compatible with CellTiter-Glo® Assays?
CellTiter-Glo® assays are compatible with virtually all mammalian cell culture models. The lysis-based mechanism works equally well for adherent cells, suspension cultures and more complex 3D model systems.
Adherent cell lines (e.g., HeLa, A549, MCF-7)
Suspension cell lines (e.g., Jurkat, K562)
Primary cells and patient-derived samples
3D spheroid and organoid cultures (see the CellTiter-Glo® 3D Assay for optimized 3D performance)
Cells grown in serum-supplemented or serum-free medium
What Are the Common Applications of the CellTiter-Glo® Assay?
The CellTiter-Glo® Assay is used across a wide range of life science research and drug discovery applications. Its high-throughput compatibility and low coefficient of variation make it a preferred choice for:
Cell viability and proliferation studies
Cytotoxicity testing—determining IC 50 values for small molecules, biologics, and ADCs
Drug screening and compound library profiling in 96-, 384-, and 1536-well formats
Cancer biology—evaluating tumor cell response to chemotherapy, targeted therapy, or radiation
Antiviral research—quantifying cytopathic effect inhibition (e.g., SARS-CoV-2 antiviral studies)
Functional genomics—measuring cell survival after RNAi knockdown or CRISPR perturbation
Immune effector assays—assessing T cell or NK cell cytotoxicity
Anan Quan, Research Assistant, Beth Israel Deaconess Medical Center .
Dr. Colleen Jonsson, Director of the Regional Biocontainment Laboratory and Director of the Institute for the Study of Host-Pathogen Systems, University of Tennessee Health Science Center .
Read more about Dr. Jonsson's work to identify antiviral agents.
