The CellTiter-Blue assay is a method for estimating the number of viable cells by measuring the metabolic activity of a cell population. It is a homogeneous assay, meaning a single reagent is added directly to the cells in culture to produce a fluorescent signal. This simple “add-incubate-measure” format reduces handling steps and makes the assay well-suited for screening many samples at once.
The Biochemical Mechanism
The CellTiter-Blue assay relies on a biochemical reaction centered around a dye called resazurin. Resazurin is a blue, cell-permeable compound that is not very fluorescent on its own. The assay reagent is a buffered solution containing a highly purified form of this dye. When added to a cell culture, resazurin can enter living cells.
Healthy, metabolically active cells constantly perform chemical reactions that create a “reducing” internal environment. This environment is characterized by a surplus of electrons. Within the cell, various enzymes, such as diaphorases, can transfer these electrons to the resazurin molecule.
This transfer of electrons biochemically reduces resazurin, converting it into a different molecule named resorufin. Unlike its precursor, resorufin is a pink-colored compound that is highly fluorescent. Nonviable cells lose their metabolic capacity and cannot perform this conversion, so they do not produce a fluorescent signal.
Assay Procedure Overview
Performing the CellTiter-Blue assay in a laboratory involves a sequence of straightforward steps. The process begins with cell plating, where cells are dispensed into the wells of a multi-well plate. These plates often contain 96 or 384 individual wells, allowing for many tests to be run simultaneously. The cells are then commonly exposed to various test compounds, such as potential drugs, for a specific duration.
Following the treatment period, the CellTiter-Blue reagent is added directly to each well containing the cells and culture medium. No cell washing or medium removal is required, which simplifies the process significantly. This single addition contains the necessary resazurin dye optimized for the viability measurement.
The plate is then placed in an incubator, typically set at 37°C, for a period that generally ranges from one to four hours. During this incubation, viable cells metabolize the resazurin and convert it into the fluorescent resorufin. The exact time needed can depend on the specific type of cells being used and their density in the wells.
Once the incubation is complete, the plate is transferred to a specialized instrument called a plate-reading fluorometer. This device measures the intensity of the fluorescent signal from each well. Although the results can also be measured by absorbance with a spectrophotometer, fluorescence is the preferred method because it provides greater sensitivity.
Data Analysis and Interpretation
To ensure the results are meaningful, the use of controls is a necessity. A positive control consists of untreated cells, which represents the baseline of 100% cell viability for that specific experiment. This sample provides the maximum signal against which all other samples are compared.
A background, or blank, control is also used. This consists of cell culture medium and the assay reagent but no cells. This measurement accounts for any intrinsic fluorescence from the medium or reagent itself, and this value is subtracted from all other readings to correct the final data.
A negative control, where cells are treated with a substance known to be toxic, confirms that the assay system can effectively detect a decrease in viability.
For comparative analysis, the fluorescence data are often normalized. The corrected reading from each experimental well is expressed as a percentage of the positive control (untreated cells). This calculation allows researchers to compare the cytotoxic effects of different compounds or concentrations, often visualized by plotting these percentages on a graph.
Common Applications and Considerations
The CellTiter-Blue assay is widely used in scientific research, primarily for cytotoxicity screening. In this application, researchers test large libraries of chemical compounds to identify substances that can kill targeted cells, such as cancer cells. This is a common step in the early phases of drug discovery. The assay is also applied in cell proliferation studies to assess how various conditions or growth factors influence the rate at which cells multiply over time.
A feature of this assay is its non-lytic nature, meaning it does not break open and kill the cells to get a reading. This allows for multiplexing, which is the practice of performing additional assays on the same plate of cells. After measuring viability with CellTiter-Blue, a researcher could, for instance, use a different assay to measure apoptosis (a specific type of cell death) in the same cell population.
When planning an experiment, the incubation time is an important factor. The optimal duration for cells to convert resazurin can differ based on the cell type and the number of cells plated per well. Therefore, some initial optimization is often required to determine the ideal incubation period that yields the most reliable and linear results.