The CellTiter-Glo assay is a rapid, homogeneous method used in biological research to determine the number of metabolically active cells in a sample. This technique offers a significant advantage because it streamlines the process into a simple addition of a single reagent to the cell culture. The assay is well-suited for automation and high-throughput applications, such as screening thousands of compounds in drug discovery, due to its speed and minimal hands-on time. It provides a simple, quantitative measure that correlates directly with cell viability, making it a routine tool for assessing cell health and proliferation.
How the Assay Measures Viability
The scientific principle behind the CellTiter-Glo assay is the quantification of intracellular Adenosine Triphosphate (ATP), a molecule present in all metabolically active cells. Because ATP is rapidly degraded upon cell death, its presence serves as a reliable marker for cell viability. The assay reagent is specifically formulated to simultaneously lyse the cell membrane, releasing the ATP into the surrounding medium, and provide the components necessary for a light-producing reaction.
This reaction is catalyzed by the enzyme luciferase, which uses the released ATP as a substrate to convert luciferin into oxyluciferin, generating bioluminescence. The amount of light generated is directly proportional to the amount of ATP released from the viable cells in the sample. A specialized instrument called a luminometer measures this light intensity, which researchers then use to determine the number of living cells. The proprietary luciferase enzyme is thermostable, resulting in a stable “glow-type” luminescence signal.
Essential Steps for Running the Protocol
Executing the CellTiter-Glo protocol begins with the proper preparation of the reagent, which typically involves reconstituting a lyophilized substrate with a proprietary buffer. This mixed reagent must be equilibrated to room temperature before use to ensure optimal reaction kinetics and consistent results across all samples. The cells to be analyzed are typically cultured in multi-well plates, such as 96- or 384-well formats, which are also brought to room temperature before the assay begins.
The protocol follows a straightforward “add-mix-measure” format. An equal volume of the prepared CellTiter-Glo Reagent is added directly to the cell culture medium in each well. A standard ratio involves adding one volume of reagent to one volume of culture medium. This single addition immediately causes cell lysis and initiates the luminescent reaction, eliminating the need for wash steps or media removal.
After the reagent addition, the plate requires thorough mixing to ensure complete cell lysis and homogenous distribution of the reaction components. This is typically achieved by placing the plate on an orbital shaker for a brief period. Following the mixing, the plate is incubated at room temperature for approximately 10 to 30 minutes, allowing the luminescent signal to stabilize before measurement. The final step is to measure the light output using a luminometer, which records the data in Relative Luminescence Units (RLU).
Common Uses and Interpretation of Results
The CellTiter-Glo assay is used extensively in drug discovery and toxicology for its application in high-throughput screening (HTS) of chemical libraries. Its speed and sensitivity allow researchers to rapidly assess the effect of thousands of compounds on cell growth and survival. This includes cytotoxicity testing, which quantifies the loss of viable cells after exposure to a potential toxin, and cell proliferation studies, which measure the increase in viable cells over time.
Interpreting the results relies on comparing the raw light signal (RLU) from treated wells to various control wells. A blank control, containing only culture medium and reagent, establishes the background signal that must be subtracted from all experimental values. A maximum viability control, consisting of untreated, healthy cells, provides the RLU value representing 100% viability, which is used to normalize the data.
The normalized data expresses the viability of treated cells as a percentage of the healthy control. A decrease in RLU in a treated well indicates a loss of viable cells, suggesting the compound is cytotoxic. Conversely, an increase in RLU suggests a growth-promoting effect in a proliferation assay.