A. Reagent Preparation
- Prepare 1X Wash Buffer by diluting 20X Wash Buffer (included in each BrdU ELISA Kit) in purified water.
- Prepare 1X detection antibody solution by diluting BrdU Detection Antibody 1:100 with Detection Antibody Diluent (green).
- Prepare 1X HRP-conjugated secondary antibody solution by diluting Anti-mouse IgG, HRP-linked Antibody 1:100 with HRP-linked Antibody Diluent (red).
- Prepare 10X BrdU solution by diluting BrdU 1:100 with cell culture medium.
B. BrdU Incorporation
- Plate cells in 96-well plate and incubate with respective test substance. Typical seed cell number is 2500–100000 cells/well depending on cell growth rate. Typical incubation time is 1–72 hr.
- Add prepared 10X BrdU solution to plate wells, for a final 1X concentration. (Example: For 100 µl medium in the plate, add 10 µl of 10X BrdU solution per well.)
- Place cells in incubator. Typical incubation time is 1–24 hr.
- Remove medium. For suspension cells, centrifuge the plate at 300 g for 10 min, then remove medium.
C. BrdU Assay
- Add 100 µl/well of the Fixing/Denaturing Solution, keep the plate at room temperature for 30 min. Remove solution.
- Add 100 µl/well prepared 1X detection antibody solution, keep plate at room temperature for 1 hr. Remove solution and wash plate 3 times with 1X Wash Buffer.
- Add 100 µl/well prepared 1X HRP-conjugated secondary antibody solution, keep plate at room temperature for 30 min. Remove the solution and wash plate 3 times with 1X Wash Buffer.
- Prepare Working Solution by mixing equal parts Luminol/Enhancer Solution and Stable Peroxide Buffer.
- Add 100 ml of the Working Solution to each well. Use a plate-based luminometer to measure Relative Light Units (RLU) at 425 nM within 1–10 min following addition of the substrate. Optimal signal intensity is achieved when read within 10 min.