A. Reagent Preparation

  1. Prepare 1X Wash Buffer by diluting 20X Wash Buffer (included in each BrdU ELISA Kit) in purified water.
  2. Prepare 1X detection antibody solution by diluting BrdU Detection Antibody 1:100 with Detection Antibody Diluent (green).
  3. Prepare 1X HRP-conjugated secondary antibody solution by diluting Anti-mouse IgG, HRP-linked Antibody 1:100 with HRP-linked Antibody Diluent (red).
  4. Prepare 10X BrdU solution by diluting BrdU 1:100 with cell culture medium.

B. BrdU Incorporation

  1. 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.
  2. 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.)
  3. Place cells in incubator. Typical incubation time is 1–24 hr.
  4. Remove medium. For suspension cells, centrifuge the plate at 300 g for 10 min, then remove medium.

C. BrdU Assay

  1. Add 100 µl/well of the Fixing/Denaturing Solution, keep the plate at room temperature for 30 min. Remove solution.
  2. 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.
  3. 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.
  4. Prepare Working Solution by mixing equal parts Luminol/Enhancer Solution and Stable Peroxide Buffer.
  5. 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.

posted October 2012

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