The Sulforhodamine B (SRB) assay is a widely used and reliable method in cell biology and toxicology. This colorimetric assay quantifies cell density and determines the cytotoxic effects of various compounds. It operates by measuring the total cellular protein content, providing a robust indication of cell number.
Mechanism of the SRB Assay
The SRB assay relies on the properties of the Sulforhodamine B dye. SRB is a bright-pink aminoxanthene dye characterized by two sulfonic acid groups, which are negatively charged. Under mildly acidic conditions, these sulfonic acid groups interact electrostatically with the positively charged basic amino acids, such as lysine and arginine, in cellular proteins.
Before staining, cells are typically fixed using trichloroacetic acid (TCA), which precipitates cellular proteins and immobilizes them onto the culture plate. The binding of SRB to these fixed cellular proteins is stoichiometric, meaning the amount of dye bound is directly proportional to the total cellular protein mass. Thus, the quantity of bound dye directly reflects the number of cells.
The SRB Assay Procedure
The SRB assay begins with Cell Seeding and Treatment. Cells are cultured in multi-well plates and allowed to adhere overnight. Following adherence, these cells are exposed to the experimental substance, such as a potential drug or chemical, for a specified duration, often 24 to 72 hours.
After the treatment period, the cells undergo Fixation by adding cold 10% trichloroacetic acid to the culture medium. This acid precipitates the cellular proteins and anchors the cells to the wells, preventing their detachment during washing. The plates are then incubated at a cold temperature, typically 4°C, for about an hour to ensure complete fixation.
Following fixation, the plates are washed with water to remove residual medium, serum proteins, and excess TCA. This prepares the fixed cells for Staining with a 0.4% (w/v) SRB solution dissolved in 1% acetic acid. The SRB solution is added to each well, and the plates are incubated at room temperature, usually for 15-30 minutes, allowing the dye to bind to the cellular proteins.
After staining, Washing is performed with 1% acetic acid to remove unbound SRB dye. This ensures that only the dye bound to the cellular proteins contributes to the final measurement, minimizing background signal. The plates are then air-dried to remove all traces of the wash solution.
Finally, for Solubilization and Measurement, the protein-bound SRB dye is dissolved using a basic solution, commonly 10 mM Trizma base. This produces a homogeneous pink solution. The absorbance of this solution is then measured at a wavelength of 510 nm using a spectrophotometer, often a plate reader. The intensity of the pink color, and the measured absorbance, directly correlates with the amount of cellular protein and, by extension, the number of cells initially present.
Applications in Cytotoxicity and Drug Screening
The SRB assay is widely used in pharmacology and cancer research, particularly for high-throughput drug screening. It allows rapid testing of thousands of chemical compounds against various cancer cell lines. This process helps identify compounds that exhibit cytotoxic effects, meaning they can kill or inhibit the growth of cancer cells.
Beyond cancer research, the assay applies to general toxicology studies. It assesses the safety profiles of diverse chemicals, environmental pollutants, or consumer products. By measuring the impact of these substances on cell viability and proliferation, the SRB assay contributes to understanding their potential harmful effects on biological systems. It provides a straightforward and quantitative readout of cellular response to external agents.
Comparison with Other Cell Viability Assays
The SRB assay offers advantages over other common cell viability methods, such as metabolic assays like the MTT, MTS, or WST assays. The main difference lies in their underlying mechanisms. The SRB assay directly measures total cellular protein biomass, which serves as a stable indicator of cell number.
Conversely, metabolic assays quantify the metabolic activity of living cells by measuring the activity of mitochondrial reductases. This distinction means that SRB is less susceptible to interference from compounds that alter cellular metabolism without directly affecting cell number or integrity. For instance, a compound that inhibits mitochondrial function but does not cause cell death would show a false positive in metabolic assays, while the SRB assay would more accurately reflect the true cell count.
Practically, the SRB assay is more cost-effective and simpler in its endpoint measurement. Fixed and stained plates can be stored for extended periods before solubilization and reading, offering flexibility in experimental design. Because the final measurement involves fixed cells, there is no risk of handling live, potentially infectious, biological material during the reading phase, contributing to laboratory safety.