Measure cell proliferation --- BrdU ELISA
This protocol is for Cell Proliferation ELISA Kit.

1. Seed 100 l (500 - 2000 cells) per well in 96 well plate (clear, flat bottom).

2. You may need to add dilution of drugs or other agents at this time or after cells have attached overnight.

3. Incubate cells for the desired time at 37 C.

4. Add 10 l BrdU labeling solution (100 M BrdU in medium) per well. Continue incubation at 37 C for 2-24 hours.

5. Remove medium, dry cells with a hair-drier for about 15 minutes or 60 C for 1 hour. Dried cells can be stored for up to one week at 4 C.

6. Add 200 l FixDenat per well and incubate for 30 minutes at 15-25 C.

7. Remove FixDenat by tapping. Add 100 l anti-BrdU-POD (1:100) per well and incubate for 30-120 minutes at 15-25 C.

8. Wash three times with 200 l washing solution (1:10). Leave the washing buffer in the plate for 1-5 minutes during the wash.

9. Remove washing buffer. Seal the clear bottom with a black adhesive foil, add 100 l substrate solution (1 B:100 A) to each well. Incubate at 15-25 C for at least 3 minutes, but no longer than 10 minutes.

10. Measure the light emission in micoplate reader within 10 minutes after adding the substrate.