BrdU Cell Proliferation ELISA Kit (colorimetric)

BrdU Cell Proliferation ELISA Kit ab126556 is a sandwich ELISA to measure BrdU incorporated cell proliferation and DNA synthesis in suspension cells and adherent cells with a sensitivity of 40 cells/well.

How the assay works

BrdU is incorporated into cells cultured in microtiter plates using the cell layer as the solid phase. During the final 2 to 24 hours of culture BrdU is added to wells of the microtiter plate. BrdU will be incorporated into the DNA of dividing cells. To enable antibody binding to the incorporated BrdU cells must be fixed, permeabilized and the DNA denatured. This is all done in one step by treatment with Fixing Solution. Detector anti-BrdU monoclonal antibody is pipetted into the wells and allowed to incubate for one hour, during which time it binds to any incorporated BrdU. Unbound antibody is washed away and horseradish peroxidase-conjugated goat anti-mouse antibody is added, which binds to the Detector Antibody. 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 reagent), the intensity of which is proportional to the amount of incorporated BrdU in the cells. The colored reaction product is quantified using a spectrophotometer.

Assay protocol summary

BrdU Cell Proliferation ELISA Kit ab126556 protocol summary:

1. Add samples and standards to wells
2. Add BrdU label. Incubate for 2-24 hours
3. Aspirate wells and add Fixing Solution. Incubate at room temperature
4. Wash each well and add detector antibody. Incubate at room temperature
5. Wash each well and add Peroxidase Conjugate. Incubate at room temperature
6. Wash each well and add TMB Substrate. Incubate at room temperature
7. Add Stop Solution to each well. Read immediately

Getting the best performance

ASSAY SENSITIVITY

The sensitivity of this BrdU cell proliferation assay was determined to be 40 cells/well using the mean signal of zero plus two standard deviations; that is, the smallest number of cells that may be distinguished from zero with 95% confidence. Using a two-hour BrdU labeling, 100 cells/well was also significantly higher than the blank control.

STORAGE

Store kit at +4°C immediately upon receipt except for the Prediluted Anti-BrdU Detector Antibody and Peroxidase Goat anti-mouse IgG (2000X) which must be stored at -20°C.

How other researchers are using this kit

BrdU Cell Proliferation ELISA Kit ab126556 has been used to study the dysregulation of RNA translation in childhood medulloblastoma (1) and effects of fatty acids on cholesterol flux and pancreatic cancer cells (2).

References: 1-John R Prensner et al. 2023, PMID: 38176414, 2-Ivar P Gladhaug et al. 2023, PMID: 37978383

Related and recommended productsOther products to measure cell proliferation include:

-ATP Assay Kit (Colorimetric/Fluorometric) ATP Assay Kit (Colorimetric/Fluorometric) ab83355
-Phalloidin-iFluor 488 Reagent Phalloidin-iFluor 488 Reagent ab176753

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