BrdU Cell Proliferation BioAssay™ Kit

Halogenated nucleotides such as the pyrimidine analog bromodeoxyuridine (BrdU) are useful for labeling nascent DNA in living cells and tissues. BrdU becomes incorporated into replicating DNA in place of thymidine and subsequent immunodetection of BrdU using specific monoclonal antibodies allows labeling of cells in S phase of the cell cycle. After pulse-labeling cells or tissues with bromodeoxyuridine, BrdU (Bu20a) Mouse mAb can be used to detect BrdU incorporated into single stranded DNA. ||The BrdU Cell Proliferation BioAssay™ Kit detects 5-bromo-2’-deoxyuridine (BrdU) incorporated into cellular DNA during cell proliferation using an anti-BrdU antibody. When cells are cultured with labeling medium that contains BrdU, this pyrimidine analog is incorporated in place of thymidine into the newly synthesized DNA of proliferating cells. After removing labeling medium, cells are fixed and the DNA is denatured with our fixing/denaturing solution. Then a BrdU mouse mAb is added to detect the incorporated BrdU (The denaturing of DNA is necessary to improve the accessibility of the incorporated BrdU to the detection antibody). Anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate TMB is added to develop color. The magnitude of the absorbance for the developed color is proportional to the quantity of BrdU incorporated into cells, which is a direct indication of cell proliferation.||The kit detects BrdU incorporation into cellular DNA during cell proliferation. The BrdU-labeled DNA has to be denatured to be detected by the BrdU Mouse mAb used in this kit. This BrdU Mouse mAb does not cross react with endogenous DNA. Depending on the cell type and the incubation time applied in the assay, 0.2-2x10e4 cells/well are sufficient for most experimental setups. ||Assay Protocol:|1. Add 100ul/well of the Fixing/Denaturing Solution, keep the plate at room temperature for 30 min. Remove solution.|2. Add 100ul/well prepared 1X detection antibody solution, keep plate at room temperature for 1 hour. Remove solution and wash plate 3 times with 1X Wash Buffer.|3. Add 100ul/well prepared 1X HRP-conjugated secondary antibody solution, keep plate at room temperature for 30 min. Remove the solution and wash plate 3 times with 1X Wash Buffer.|4. Add 100ul TMB Substrate.|5. Incubate for 30 min at room temperature. |Note: Watch the color change as it may be necessary to stop the reaction prior to the standard development time of 30 min.|6. Add 100ul Stop Solution.|7. Read absorbance at 450nm (For optimal readings, read the plate within 30 min of adding Stop Solution).||Kit Components:|1. BrdU (1000X), 1x150ul, -20°C|2. Fixing/denaturing Solution, 2x25ml, RT |3. BrdU Detection antibody (100X), 1x500ul, -20°C |4. Anti-mouse IgG, HRP-Linked Antibody: 1x500ul, -20°C|5. Detection Antibody Diluent, 1x50ml, 4°C|6. HRP Diluent, 1x50ml, 4°C|7. TMB Substrate, 1x50ml, 4°C|8. Stop Solution: 2x25ml, 4°C|9. Wash Buffer (20X),1x50ml, 4°C||Storage and Stability:|This kit contains mixed storage components. Please store this entire kit at -20C for long term storage. All components in this kit are stable for 12 months after receipt when stored at the recommended temperature and left unused.