BrdU Cell Proliferation Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- 3×103 cells/well in a 96-well plate were seeded for an assay.	- After 24 h or culture, for another 2h cells were incubated with BrdU. - anti-BrdU monoclonal antibody isincubated for 1 hr.

Upstream tips
- 3×103 cells/well in a 96-well plate were seeded for an assay.
Protocol tips
- After 24 h or culture, for another 2h cells were incubated with BrdU. - anti-BrdU monoclonal antibody isincubated for 1 hr.

Publication protocol

Cell proliferation was also assessed by 5′-bromo-2′-deoxyuridine (BrdU) incorporation. The BrdU Assay kit (Millipore) was used according to the manufacturer’s recommendations. Cells were seeded at a density of 3×103 cells/well in a 96-well plate. Following 24 hr of culture, BrdU was added and allowed to accrue within proliferating cells for 2 hr. The cells were then fixed at room temperature for 30 min followed by incubation with an anti-BrdU monoclonal antibody for 1 hr. The cells were then incubated at room temperature with a secondary antibody conjugated with peroxidase. The substrate solution tetramethylbenzindine (TMB) was added with incubation for 30 min in the dark at room temperature. The staining reaction was terminated with a 2.5 N sulfuric acid solution and the plate read with a spectrophotometer at 450 nm.

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