Description: This Bu20a monoclonal antibody reacts with 5-bromodeoxyuridine (BrdU). BrdU is a derivative of uridine that can be incorporated into DNA in place of thymidine during the S-phase of the cell cycle. Anti-BrdU can then be used to identify cells that have undergone DNA synthesis during BrdU treatment. For staining for flow cytometric analysis, we recommend the use of the BrdU Staining Buffer Set (cat. 00-5525) and protocol. Applications Reported: This Bu20A antibody has been reported for use in intracellular staining followed by flow cytometric analysis and immunohistology staining of frozen tissue sections. Applications Tested: This Bu20A antibody has been tested by flow cytometric analysis of BrdU-labeled mouse splenocytes. This can be used at less than or equal to 1 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. BrdU labeling and staining with anti-BrdU antibody:1. Label dividing cells with 10 µM BrdU for 45 min at 37°C.2. Following the incubation, harvest the cells and wash once with 1X PBS.3. Stain surface molecules according to the Surface Staining Protocol.4. Wash in cold Flow Cytometry Staining Buffer or 1X PBS.5.Resuspend the cell pellet by pulse vortexing. Then add 1 mL of freshly prepared Foxp3 Fixation/Permeabilization solution (cat. 00-5521) to each sample. pulse vortex again.6. Incubate for 30 to 60 minutes at 2-8°C in the dark.7. Wash once by adding 2 mL 1X Permeabilization Buffer (made from 10X Permeabilization Buffer included in the Foxp3/Transcription Factor Staining Buffer Set) followed by centrifugation. Decant the supernatant.8. Resuspend the cell pellet with 100 µL Flow Cytometry Staining Buffer containing 30 µg of DNase I.9. Incubate for 1 hr at 37°C and then wash.10. Stain cells with anti-BrdU antibody for 30 min to 1 hr and then wash.10. If necessary, stain with secondary antibody for 30 min and then wash.12. Analyze the samples. Purity: Greater than 90%, as determined by SDS-PAGE. Aggregation: Less than 10%, as determined by HPLC. Filtration: 0.2 µm post-manufacturing filtered. Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) is a synthetic nucleoside that is an analogue of thymidine. BrdU is commonly used in the detection of proliferating cells in living tissues, and can be incorporated into the newly synthesized DNA of replicating cells (during the S phase of the cell cycle), substituting for thymidine during DNA replication. Antibodies specific for BrdU can then be used to detect the incorporated chemical, thus indicating cells that were actively replicating their DNA. Binding of the antibody requires denaturation of the DNA, usually by exposing the cells to acid or heat.
|Flow Cytometry, Immunohistochemistry (Frozen)|
|PBS with 0.09% sodium azide; pH 7.2|
For Research Use Only.
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