Section7:Types of Tissue Culture Assays

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Contents 1 Types of Tissue Culture Assays 2 Proliferation Assays 3 Apoptosis Assays 4 Gene Expression 5 Activation Assays

Types of Tissue Culture Assays

Listed below are types of cell-based assays frequently used as primary, secondary or profiling assays for screening compounds to find new chemical entities.

Proliferation Assays

Used to measure cell growth over a period of time and the effects of compounds on the cell growth.

1. Thymidine uptake is measured by using 96 well plates coated with scintillant or using filter binding methods. Plates containing scintillant detect the thymidine taken up by the cells by using 14C or 3H.
2. WST reagents: A colorimetric assay for the quantification of cell proliferation and cell viability, based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells (a soluble version of MTT or MTX)
3. Alamar Blue™: A colorimetric assay
4. There are several non-radioactive, fluorescence-based assays that are not dependent on cellular metabolism. The fluorescent dye binds nucleic acids and the fluorescesences can then be measured quantitatively or qualitatively. Propidium Iodide, Hoechst or other live dyes can be added to cells. The fluorescence can than be detected using various high content imaging instruments. The cell number can then be quantitated based on the fluorescence. DNA content can also be quantitated using the tools available in the imaging instruments.
5. Chemiluminescent, non-separation assay kits for the determination of viable cell numbers are also available for either proliferation or cytotoxicity assays.

Apoptosis Assays

Used to measure the various stages of cell death referred to as early, intermediate and terminal apoptosis. undergoing apoptosis. In just one of many scenarios of apoptosis, the process is triggered by another neighboring cell; the dying cell eventually transmits signals that tell the phagocytes, which are a part of the immune system, to engulf it. There are numerous assays and markers available on the market to determine if the cells are undergoing apoptosis and where in the apoptosis pathway they are. Some examples of these markers are listed below.

1. Annexin V staining
2. Tunnel staining
3. Caspase activity
4. Nuclear Fragmentation using microscopes, or other imaging platforms

Gene Expression

1. Transient transfection of a gene of interest into a cell using various reporter systems. There are numerous transient transfection reagents and protocols available that have been optimized for various cell lines and promoters.
2. Stable transfection of a gene, is where the gene of interest is stably integrated into the cell. Stable transfections can take a lengthy period of time to be generated.

Activation Assays

Used to measure changes in the cellular mechanisms following treatment with compounds.

1. A. Measurements of intracellular second messengers: calcium flux, cAMP. There are numerous assay formats available for measuring cAMP including ELISA’s, radioactive formats, homogenous formats including FRET assay formats.and alpha screen.
2. Uptake of various cellular components measured by using radioactive ligands.
3. Activation of metabolic pathways:measurement of phosphorylation events intracellular or intranuclear.