BrdU Cell Proliferation Assay Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- 1 × 104 cells were plated for 24h - Allowed to serum starve for another 24h before different kinds of medias were added. - For another 24-72 h different kinds of chemicals, recombinant proteins or neutralizing antibodies were added.	- Cells were treated with BrdU for 24 h.

Upstream tips
- 1 × 104 cells were plated for 24h - Allowed to serum starve for another 24h before different kinds of medias were added. - For another 24-72 h different kinds of chemicals, recombinant proteins or neutralizing antibodies were added.
Protocol tips
- Cells were treated with BrdU for 24 h.

Publication protocol

Briefly, 1 × 104 cells were plated into 96-well plate for 24 h and serum-starved for additional 24 h, before treated with complete media, control media (media containing 1% FBS) or fibroblast conditioned media (1.0 μg/μl) with or without chemicals, recombinant proteins or neutralizing antibodies (BioLegend, CA, USA) for subsequent 24-72 h. At the end of treatment, MTT solution (5 mg/ml) was added into each well, followed by 4 h incubation at 37°C. The formazan crystals were dissolved with sodium dodecyl sulfate before reading the absorbance using CHAMELEON™V plate reader (Hidex, Finland) at 570 nm with reference wavelength of 630 nm.

To measure cell proliferation, we determined BrdU incorporation into the cellular DNA using an ELISA-based approach (Cell Signaling Technology, MA, USA). Cells were seeded and treated as described above, and at the end of treatment, BrdU solution was added for 24 h. Following fixation and denaturation, the cells were labeled with antibodies and washed before added with TMB substrate. Color development was terminated with a STOP solution, and measured at 450 nm using CHAMELEON™V plate reader. In addition, the cells were also stained with 0.04% trypan blue to count for number of viable cells, using neubauer improved hemocytometer (Sigma Aldrich).

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