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5′-bromo-2′-deoxyuridine (BrdU) is a synthetic analogue of the nucleoside thymidine, in which the 5-methyl group is substituted by bromine. It is commonly used for the detection of proliferating cells via its incorporation into newly synthesized DNA; replicating cells can subsequently be identified through the use of anti-BrdU antibodies.
BrdU has been used for decades and has extremely wide utility. Applications in which it has been cited include stem cell research, studies into the effects of test compounds on cell cycle progression, monitoring of developmental progress during embryogenesis and investigation of neurogenesis, to name just a few.
BrdU labeling can be performed for cultured cells, with analysis via microscopy, flow cytometry or ELISA. Alternatively, BrdU may be administered to animals, with analysis via immunohistochemistry (IHC) following sacrifice. Irrespective of the technique, successful antibody-based detection of BrdU requires that a DNA denaturing step is included in the staining protocol to allow the antibody access to its target. Hydrochloric acid (HCl) is typically used for this purpose although, similarly to every step of an assay, the concentration and incubation time require careful optimization.
Exalpha Biologicals offers BrdU reagent, high quality anti-BrdU antibodies, and complete kits for BrdU-based detection of cell proliferation.
BrdU proliferation ELISA kits – colorimetric or chemiluminescent readout
Exalpha Biologicals’ BrdU proliferation ELISA kits rely on the incorporation of BrdU into adherent cells cultured in microtiter plates. BrdU is added to the growth media during the final stages of culture, following which a fixing solution is used to fix and permeabilize the cells, and denature the DNA, all in a single step. The cells are then incubated with an anti-BrdU antibody prior to detection with an HRP-conjugated antibody and TMB substrate (colorimetric readout) or diacylhydrazide substrate (chemiluminescence).
BrdU proliferation immunocytochemistry / IHC kits – colorimetric or fluorescent readout
Exalpha Biologicals’ BrdU proliferation immunocytochemistry / IHC kits are suitable for use with paraffin-embedded tissue sections or with cultured cells. Visual confirmation that BrdU has been incorporated into proliferating cells in vitro or in vivo is achieved using a biotinylated anti-BrdU antibody followed by detection with a streptavidin-HRP conjugate and DAB substrate (colorimetric readout) or a streptavidin-fluorophore conjugate (fluorescent readout). Each kit contains sufficient material to process fifty slides, and includes five control slides (intestinal tissue from a mouse injected with BrdU). Two options are available for the fluorescent readout; a microscope equipped with an excitation filter of 493nm is required for FL488, while an excitation filter of 556nm is necessary for FL549.