BrdU Cell Proliferation Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells were seeded at a cell density of 1×105 cells/ml.	- Seeded cells were treated with insulin (25 nM) for 24h. - Cells were incubated with BrdU for 24 h.	- The absorbance was measured at a dual wavelength of 450–540 nm

Upstream tips
- Cells were seeded at a cell density of 1×105 cells/ml.
Protocol tips
- Seeded cells were treated with insulin (25 nM) for 24h. - Cells were incubated with BrdU for 24 h.
Downstream tips
- The absorbance was measured at a dual wavelength of 450–540 nm

Publication protocol

The BrdU cell proliferation assay is a non-isotopic immunoassay for the quantification of BrdU incorporation into newly synthesized DNA of actively proliferating cells. MCF-7 and MDA-MB-231 cells were seeded into a 96-well cell culture plate at a cell density of 1×105 cells/ml. Cells were allowed to attach and were treated with insulin (25 nM) for 24 h in normal- as well as in high-glucose conditions. The cells were then incubated with BrdU for 24 h and the assay was performed further according to the manufacturer's instructions. The absorbance was measured at a dual wavelength of 450–540 nm using a Flexstation III spectrophotometer (Molecular Devices). The result was calculated as percentage of cell proliferation compared with normal glucose control

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