Cell-Based Assays

The current state of cells and their functions during certain conditions is central to biomedical research and clinical drug development. In response to chemical or environmental stimuli, researchers need to assess responses that occur at the cellular level. Cell-based assays are a collection of biochemical techniques used to evaluate and characterize the state, function, or activity of cells and tissues. Outside of disease research, cellular assays can be used in routine measurements of cell health and viability. Such assays can also enable cell studies in development, metabolism, and function. A diverse variety of cellular assays are available, each focusing on a particular method of detection. We have curated a diverse catalog of cell-based assays to help support life science researchers.

Cell Viability Assays

Cell viability is a measure of survivability; it is a method of assessing the ability of cells, tissues, and organs to survive or recover. While living cells can be observed visually, cell viability assays use biochemical methods to provide a quantifiable measure of survivability. Many viability assays look at active cell metabolism through the activity of metabolic enyzmes.

• WST Assay - This test operates on the colorimetric conversion of water-soluble tetrazolium (WST) to dark red formazan by active mitochondrial reductases. WST does not require a solubilization step and is less toxic to cells.
• MTT Assay - This involves the conversion of the yellow tetrazolium MTT to an insoluble purple formazan by active NAD(P)H-dependent cellular oxidoreductases. Cell viability is proportional to the intensity of color produced by the reaction.
• XTT Assay - This is a colorimetric assay that reduces the yellow tetrazolium XTT to an orange formazan dye in the presence of metabolically active cellular dehydrogenases.
• MTS Assay - Like the MTT assay, the MTS assay operates on the conversion of a tetrazolium salt to a colored product. However, reagents in the MTS assay can be added directly to cell media, saving additional steps.
• Live/Dead Assay - Dyes are used for the simultaneous evaluation of live and dead cells within a population. Often, a fluorescent reagent is used for a two-color distinction of the live and dead cells.

Cell Proliferation Assays

Cell proliferation, like cell viability, is an important aspect of cell health that indicates actively dividing or replicating cells. However, not all viable cells are guaranteed to be proliferating. Thus, cell proliferation assays help researchers identify the subset of proliferative cells within a viable cell population. Methods to assess cell proliferation can include visualizing active DNA synthesis and using dyes to track cell division.

• BrdU Assay - Bromodeoxyuridine (BrdU) is a thymidine analog that can be incorporated in cellular DNA in replication. Using BrdU-specific antibodies, actively dividing cells can be identified and counted.
• Cell Tracking - Dyes such as CFDA-SE and CFSE are taken up by cells for a long period. As the cells divide, the intensity of the stain is halved. This allows tracking of succeeding cell generations.

Apoptosis Assays

Apoptosis, or programmed cell death, is an important cellular process as apoptotic pathways in disease are often targets for drug therapy. Cells undergoing apoptosis exhibit characteristic changes, including DNA fragmentation, plasma membrane reorganization, blebbing, and shrinkage. A variety of biochemical apoptosis assays and staining kits can be used to study the effects or progression of cell death in cells and tissues.

• Caspase Assay - Caspases are a family of proteases notable for their role in apoptotic signaling pathways. Assays for caspase activity generally work by adding a specific peptide substrate to live cells or cell lysates.
• Annexin V Staining - Annexin V is a protein that binds to phosphatidylserines exposed in the extracellular membrane, a hallmark of apoptosis. Labeled annexin V is commonly used to stain and identify apoptotic cells.
• TUNEL Assay - Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is a method of detecting cellular DNA fragmentation. The TdT transferase finds nicked DNA and adds dUTP, which can be labeled for detection.
• Comet Assay - Electrophoresis is used to separate damaged DNA from intact DNA, producing a shape that resembles the tail of a comet. The shape of the comet provides a measure of cellular DNA damage.
• Lysosome Staining - Lysosomes, which contain cathepsin proteases, are linked to apoptosis and other forms of cell death. Lysosome staining provides a method of assessing lysosome structure and function.
• Cathepsin Assay - Cathepsins are proteases that not only play a central role in cell death, but are also linked to apoptotic processes. These assays are used to measure the activity of specific cathepsin family members.

Cytotoxicity Assays

Cellular cytotoxicity is the loss of cellular structure and function in response to unfavorable conditions, such as stress or cytotoxic compounds. Hallmarks of cytotoxicity include cytoplasmic leakage of biomarkers and cell membrane damage. Specific methods of cytotoxicity assays are listed below.

• LDH Assay - Leakage of the metabolic enzyme lactate dehydrogenase (LDH) from the cytoplasm is a common marker of cytotoxicity. LDH assays measure the conversion of lactate to pyruvate as an indicator of LDH activity.
• Live/Dead Staining - Using a combination of dyes that are permeable and impermeable to membranes allow researchers to distinguish between living and dead cells.

Assays for Cellular Metabolism and Function

In addition to cell health, researchers may also wish to evaluate the functions of cells. Cells are alive, after all, and are able to move and metabolize compounds. Assays are commercially available that are designed to measure specific cellular functions.

• ATP Assay - ATP acts as the main energy carrier in living cells and can be used as a proxy for active cellular metabolism. Some assays use luciferase to generate luminescence from ATP. Other assays utilize the phosphorylation of glycerol for colorimetric or fluorometric detection.
• Glucose Uptake Assay - 2-deoxyglucose (2-DG), a structural analogue of glucose, is converted by cells to 2-DG-6-phosphate (2-DG6P). As 2-DG6P cannot be further metabolized, its accumulation serves as a measure of glucose uptake.
• Invasion Assay - Cell invasion assays use membranes coated with substances such as collagen or basement membrane, which allow cells to pass through in response to stimulants or inhibitors.
• Migration Assay - Cells migrate in response to stimuli, including chemical and mechanical signals. Migration and chemotaxis assays are used to distinguish between migratory from non-migratory cells.
• Phagocytosis Assay - Many of these kits use inactive labeled pathogens to induce phagocytosis. Such kits can be used to study activators and inhibitors of phagocytosis and its related pathways.