Since BrdU only incorporates into newly synthesized DNA, the presence of non-proliferating cells and dead cell debris will not affect the results as they would in methods that involve quantifying total biomass or total viable cells. After BrdU labeling, medium can be removed by tapping off or suction.
After removal of the fixative, the procedures are more straightforward. Incorporated BrdU is then detected by colorimetric immunoassay. This protocol is to be used to incorporate and detect BrdU in murine plasma cells.
Incubate cells for 20 min at room temperature. In addition to evaluation of cell proliferation, information such as cell number, morphology and analysis of cellular antigens can be obtained from a single culture.
This is all done in one step by treatment with Fixing Solution.
Thorough removal of this solution is a little difficult, however. 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 reagentthe intensity of which is proportional to the amount of incorporated BrdU in the cells.
Incubate cells for 10 min on ice. During the final 2 to 24 hours of culture BrdU is added to wells of the microtiter plate. Incubate for 30 min on ice for fix cells.
Multiple steps standard assay Product overview ab involves incorporation of BrdU into cells cultured in microtiter plates using the cell layer as the solid phase. In addition, the high flexibility of the protocol and wide dynamic range of the assay let me design my experiment easily.
Sacrifice the mice and harvest the spleen. Discard supernatant and resuspend cells with 20 ml of DMEM and count cells. The above antibodies have been tested by the author and may be substituted with the antibodies desired by users. The water needs to be changed daily and be wrapped in aluminum foil to avoid light.
Unbound antibody is washed away and horseradish peroxidase-conjugated goat anti-mouse antibody is added, which binds to the Detector Antibody. The working BrdU labeling solution can be added directly to the treated cell culture.
BrdU Sigma-Aldrich, catalog number: Create single cell suspension by gently smashing spleen pieces with the frosted surface of a pair of microscope slides in 5 ml of DMEM. Incubate cells at room temperature for 15 min. This kit is quite handy to use as it provides all the reagents required for the assay.
A wide range of initial seeding densities, drug treatment durations and BrdU labeling times can be accommodated. Resuspend cells with 5 ml of 1x ammonium chloride lysing solution see Recipes and incubate on ice for 5 min. On the other hand, directly counting cells would be labor-intensive and impractical if the sample number is very large.
The resultant assay is sensitive, rapid, easy to perform and applicable to high sample throughput. Tested applications Suitable for:BrdU Cell Proliferation Assay Kit Protocol A.
Prepare 1X Wash Buffer by diluting 20X Wash Buffer (included in each BrdU ELISA Kit) in purified water. BrdU (Bromodeoxyuridine) is a commonly used tool for cell proliferation and cell cycle studies.
We look at its structure and use and provide.
BrdU Cell Proliferation Protocol. Specific for product: BrdU Cell Proliferation Assay Kit # A. Reagent Preparation. Prepare 1X Wash Buffer by diluting 20X Wash Buffer (included in each BrdU ELISA Kit) in purified water.
BrdU (Bromodeoxyuridine or 5-bromo-2’-deoxyuridine) is a synthetic nucleoside that is incorporated into DNA by proliferating cells. This protocol is to be used to incorporate and detect BrdU in murine plasma cells. The plasma cells described in this protocol are formed spontaneously in autoimmune mice (NZB/W mice).
Modifications are most likely. Protocol describing how to BrdU label HeLa cells and detect the incorporated BrdU with the help of rabbit and mouse anti-BrdU antibodies. BrdU Labeling of HeLa Cells Followed by Immunostaining with Mouse Anti-BrdU Antibody (clone Bu20a).
Abcam’s BrdU Cell Proliferation ELISA Kit (colorimetric) in vitro ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for accurate Review the protocol contain cells but will not receive the BrdU reagent (assay background).Download