5-Bromo-2′-deoxy-uridine Labeling and Detection Kit III
ROCHE/11444611001 - sufficient for ≤1,000 tests, kit of 1 (8 components), suitable for ELISA, 10000 (Nuclease activity (vial 4))
Synonym: 5-BrdU; 5-
Product Type: Chemical
Principle of the BrdU Labeling and Detection Kit III (POD).
Comparison of the sensitivity of the BrdU and [3H]-thymidine methods in measuring proliferation, as shown in hEGF (left figure) and mIL-6 (right figure) stimulation assays. Left: Proliferation of AKR-2B cells (mouse fibroblast cell line) in response to recombinant human epidermal growth factor (hEGF). Proliferation was measured with either the BrdU Labeling and Detection Kit III (red) or the [3 H]-thymidine method (blue). Right: Proliferation of 7TD1 cells (mouse-mouse hybridoma) in response to recombinant mouse interleukin-6 (mIL-6). Proliferation was measured with either the BrdU Labeling and Detection Kit III (red) or the [3 H]-thymidine method (blue). Conclusion: In both cases, the cell proliferation assay with the nonradioactive BrdU kit was just as sensitive as the radioactive thymidine assay.
| manufacturer/tradename | Roche |
| packaging | kit of 1 (8 components) |
| Quality Level | 100  |
| shipped in | wet ice |
| specific activity | 10000 (Nuclease activity (vial 4)) |
| storage temp. | 2-8°C |
| technique(s) | ELISA: suitable |
| usage | sufficient for ≤1,000 tests |
| Application: | The kit is used for the quantitative determination of BrdU incorporated into cellular DNA using a 96-well microplate cell ELISA format. 5-Bromo-2′-deoxy-uridine Labeling and Detection Kit III has been used in cell proliferation assay, BrdU assay and proliferation assay. |
| Features and Benefits: | • Safer, since the kit does not use radioisotopes. • Accurate, since results generated with this assay strongly correlate to those obtained with the [3 H]-thymidine method. (See Figure 2 below.) • Sensitive. This assay and the [3 H]-thymidine assay are equally sensitive. (See Figure 2 below.) • Fast, since results can be read with a multi-well ELISA reader, allowing simultaneous processsing of a large number of samples • Easy, since the assay uses a standard cell ELISA protocol. • Economical, since the assay requires no expensive equipment or additional reagents (e.g ., scintillation fluid). |
| General description: | Proliferation in cell populations may be studied by incorporating the radioisotope [3H]-thymidine into cellular DNA. The amount of radioactive thymidine incorporated is determined by scintillation counting. Alternatively , 5-bromo-2′-deoxy-uridine may be incorporated into cellular DNA. The amount of BrdU incorporated is determined by a standard ELISA protocol, which involves "tagging" the incorporated nucleotide with an anti-BrdU antibody. 96-well microplate cell ELISA for the detection of 5-bromo-2′-deoxy-uridine (BrdU) incorporated into cellular DNA. Nonradioactive alternative for [3H]-thymidine based DNA synthesis and cell proliferation assays. Contents • BrdU Labeling Reagent, 1,000x concentrated • Washing Buffer concentrate, 10x concentrated • Incubation Buffer • Nucleases • Anti-BrdU-POD, Fab fragments • Substrate Buffer • ABTS Substrate • Substrate Enhancer |
| Other Notes: | For life science research only. Not for use in diagnostic procedures. |
| Packaging: | 1 kit containing 8 components. |
| Preparation Note: | Working solution: BrdU labeling solution Dilute BrdU labeling reagent 1 : 90 with sterile PBS or culture medium (resulting concentration: 111 μM BrdU) [e.g., for one 96-well microplate containing 100 μl medium per well, dilute 12 μl BrdU labeling reagent with 1.068 ml sterile PBS]. Note: The BrdU labeling solution should be prepared freshly before use. Washing buffer Dilute washing buffer concentrate (10x) 1 : 10 with double-dist. water [e.g., for one 96-well microplate dilute 9 ml washing buffer concentrate (10x) with 81 ml double-dist. water]. Note: If precipitates in Washing buffer, 10x conc. are visible, please incubate the bottle for 10 minutes at 37 °C in a water bath before you prepare Solution II. Washing buffer is used to:• Prepare the anti-BrdU-peroxidase, working solution • Wash cells after incubation with anti-BrdU-peroxidase Note: For all other washing steps use PBS or culture medium containing 10% serum [e.g., FCS (fetal calf serum)] to obtain reliable results. Incubation buffer • Ready-to-use • Used to dilute the nucleases Nucleases, stock solution Reconstitute the nucleases in 1.3 ml double-dist. water containing 50% glycerol (w/v). Nucleases, working solution Dilute nucleases, stock solution, 1 : 100 with incubation buffer (e.g., for one 96-well microplate dilute 100 μl nucleases, stock solution, with 9.9 ml incubation buffer). Anti-BrdU-peroxidase, Fab fragments, stock solution Dissolve anti-BrdU-peroxidase, Fab fragments in 1.25 ml double-dist. water (final concentration: 20 U/ml). Anti-BrdU-peroxidase, Fab fragments, working solution Prepare anti-BrdU-peroxidase, Fab fragments, working solution shortly before use. Dilute anti-BrdU-peroxidase, Fab fragments, stock solution 1 : 100 with washing buffer supplemented with 10 mg/ml BSA (bovine serum albumin), [e.g., for one 96-well microplate dilute 100 μl anti-BrdU-peroxidase, Fab fragments, stock solution, with 9.9 ml PBS and BSA (final concentration: 200 mU/ml)]. Peroxidase substrate Dissolve the ABTS powder in substrate buffer and stir at 15 to 25 °C to obtain a clear solution. Peroxidase substrate containing substrate enhancer If a low signal is expected, take an appropriate aliquot of substrate solution and add substrate enhancer , 1 mg/ml and dissolve by stirring for 15 minutes at 15 to 25 °C (e.g., for one 96-well microplate dissolve 10 mg substrate enhancer in 10 ml peroxidase substrate) Note: The substrate solution containing substrate enhancer is stable for only 4 hours and should, therefore, be freshly prepared before use. Storage conditions (working solution): BrdU labeling reagent The undiluted BrdU labeling reagent (1000x) is stable at 2 to 8 °C for 6 months. It is stable stored in aliquots at -15 to -25 °C. Washing buffer Stable at 2 to 8 °C for 3 months. Incubation buffer Stable at 2 to 8 °C until the expiration date printed on the label Nucleases, stock solution Stable at -15 to -25 °C for 6 months. Nucleases, working solution must be prepared shortly before use. Anti-BrdU-Peroxidase, Fab fragments, stock solution Stable at 2 to 8 °C for 6 months. For long term storage it is recommended to store the solution in aliquots at -15 to -25 °C. Anti-BrdU-Peroxidase, Fab fragments, working solution must be prepared freshly before use. Peroxidase substrate Stable at 2 to 8 °C for 2 months when stored protected from light. Peroxidase substrate containing substrate enhancer must be prepared freshly before use |
| Principle: | Cells cultured in a 96-well microplate are incubated with BrdU (see Performance). The labeled cells are fixed with ethanol. Prior to incubation with a monoclonal antibody to BrdU, DNA is partially digested with nucleases to allow the antibody to access BrdU. Next, the anti-BrdU antibody [labeled with peroxidase (POD)] is added. Finally, the POD substrate ABTS is added. POD catalyzes the cleavage of ABTS, producing a colored reaction product. The absorbance of the samples (at approximately 405 nm) is determined with a standard microplate (ELISA) reader. |
| Specificity: | Anti-BrdU-peroxidase, Fab fragments, specifically bind to 5-bromo-2′-deoxy-uridine incorporated into DNA. It shows no cross-reactivity with any endogenous cellular components, such as thymidine or uridine. |
| Symbol |  GHS07 |
| Signal word | Warning |
| Hazard statements | H317 - H319 - H412 |
| Precautionary statements | P261 - P273 - P280 - P333 + P313 - P337 + P313 - P362 + P364 |
| RIDADR | NONH for all modes of transport |
| WGK Germany | WGK 2 |
| Flash Point(F) | does not flash |
| Flash Point(C) | does not flash |
| activity | specific activity: 10000 (Nuclease activity (vial 4)) |
| Storage Temp. | 2-8°C |
| UNSPSC | 41116133 |
| Components | BrdU Labeling Reagent 1,000x concentrated; Washing Buffer concentrate 10x concentrated; Incubation Buffer; Nucleases; Anti-BrdU-peroxidase antibody, Fab fragments; Substrate Buffer; ABTS Substrate; Substrate Enhancer |