Description
Principle of the assay: Proliferation Assay involves incorporation of BrdU into cells cultured in microtiter plates using the cell layer as the solid phase. During the fi nal 2 to 24 hours of culture BrdU is added to wells of the microtiter plate. BrdU will be incorporated into the DNA of dividing cells. To enable antibody binding to the incorporated BrdU cells must be fi xed, permeabilized and the DNA denatured. This is all done in one step by treatment with Fixing Solution. Detector anti-BrdU monoclonal antibody is pipetted into the wells and allowed to incubate for one hour, during which time it binds to any incorporated BrdU. Unbound antibody is washed away and horseradish peroxidase-conjugated goat anti-mouse antibody is added, which binds to the Detector Antibody. The horseradish peroxidase catalyzes the conversion of the chromogenic substrate tetra-methylbenzidine (TMB) from a colorless solution to a blue solution (or yellow after the addition of stopping reagent), the intensity of which is proportional to the amount of incorporated BrdU in the cells. The colored reaction product is quantifi ed using a spectrophotometer.
Background: Evaluation of cell cycle progression is essentialfor investigations in many scientifi c fi elds. Measurement of [3H] thymidine incorporation as cells enter S phase has long been the traditional method for the detection of cell proliferation. Subsequent quantifi cation of [3H] thymidine is performed by scintillation counting or autoradiography. This technology is slow, labor intensive and has several limitations including the handling and disposal of radioisotopes and the necessity of expensive equipment. A well-established alternative to [3H] thymidine uptake signal to noise ratios are obtained when longer BrdU labeling times are used. Dilute the 500X concentrated stock 1:500 by adding 8 mul of BrdU stock to 4 mls of cell media. Pipette 20 mul of the diluted BrdU label to the appropriate wells. Reminder: a series of wells should be set aside that do NOT receive the BrdU label (- BrdU control for determining assay background). Incubate the assay 2-24 hours