Cell Proliferation ELISA, BrdU

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- 100 ul Fixative/Denaturing solution is added and incubate for 30 minutes at room temperature. 100 μl of Substarte solution is added to each well and the color reaction is stopped after 10 minutes with 25 μl 1 M H2SO4.	- Incase of high background reduce cell concentartion.

Protocol tips
- 100 ul Fixative/Denaturing solution is added and incubate for 30 minutes at room temperature. 100 μl of Substarte solution is added to each well and the color reaction is stopped after 10 minutes with 25 μl 1 M H2SO4.
Downstream tips
- Incase of high background reduce cell concentartion.

Publication protocol

The cell proliferation ELISA, BrdU kit (Roche) was used according to the manufacturer’s instructions. Cells were seeded into 96-well plates (5 × 103 cells / well) and treatments were added the next day. After each treatment, cells were labeled with a 10 mM BrdU solution (5′bromo-2′deoxyuridine) in culture medium and incubated for an additional 48 h at 37°C. The medium was then replaced with 100 ul Fixative/Denaturing solution and incubate for 30 minutes at room temperature. Anti-BrdU Antibody in 1:1000 dilutions was added to each well and the plates were incubated at room temperature for 1 h. After three washing steps, peroxidase-substrate color development solution (100 μl) was added to each well and the color reaction was stopped after 10 minutes with 25 μl 1 M H2SO4. Absorbance of samples was measured at 370 nm (reference wavelength 492 nm).

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