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DNA synthesis–based cell proliferation assay

Cell proliferation can be measured with the thymidine analog BrdU (5-bromo-2’-deoxyuridine) following its incorporation into newly synthesized DNA and its subsequent detection with an anti-BrdU antibody.

This protocol can be used for:

  • Detecting DNA synthesis using a fluorescence microscope

This protocol should not be used for:

  • Flow cytometry

You will need the following for this protocol:

  • Proliferating adherent cells in an appropriate vessel for imaging
  • BrdU (5-Bromo-2´-Deoxyuridine) (Cat. No. B23151)
  • Anti-BrdU monoclonal antibody (such as Cat. No. B35130)
  • Alexa Fluor®–labeled secondary antibody (such as Cat. No. A-11001)
  • Complete tissue culture medium, such as DMEM
  • PBS or similar wash solution (Cat. No. 14040141)
  • Fixative (i.e., 3.7% formaldehyde in PBS)
  • Permeabilization buffer (0.1% Triton® X-100 in PBS)
  • 12 N HCl
  • Phosphate/citric acid buffer, pH 7.4 (182 mL of 0.2 M Na2HPO4 + 18 mL 0.1 M citric acid)
  • Antibody stain solution (PBS/0.1% Triton/5% normal goat serum)
  • Ice bucket
  • Fluorescence microscope

Protocol

Prepare stock solutions

  1. Dissolve 100 mg BrdU in 32.5 mL anhydrous DMSO (10 mM stock solution)
  2. Dilute 10 µL of this stock solution in 10 mL of 37°C tissue culture medium to make a 10 µM labeling solution 

Label cells with BrdU

  1. Culture cells in appropriate vessel for microscopy 
  2. Remove culture medium from cells and replace with BrdU labeling solution 
  3. Incubate cells at 37°C for 2 hours
  4. Remove labeling solution and wash two times with PBS
  5. Wash with PBS (3 times, 2 minutes each)

Fix, permeabilize, and acid-wash

  1. Remove PBS and add 1 mL of 3.7% formaldehyde in PBS to each well
  2. Incubate for 15 minutes at room temperature
  3. Wash with PBS (3 times, 2 minutes each)
  4. Remove PBS and add 1 mL of Triton® X-100 permeabilization buffer to each well
  5. Incubate for 20 minutes at room temperature
  6. Remove permeabilization buffer and add 1 mL of 1N HCl
  7. Incubate 10 minutes on ice
  8. Remove this solution and add 1 mL of 2N HCl
  9. Incubate 10 minutes at room temperature
  10. Add 1 mL phosphate/citric acid buffer, pH 7.4
  11. Incubate 10 minutes at room temperature
  12. Wash with Triton® X-100 permeabilization buffer (3 times, 2 minutes each)

Detect incorporated BrdU

  1. Remove this solution and add 1 mL of antibody staining buffer
  2. Add anti-BrdU primary antibody
  3. Incubate overnight at room temperature
  4. Wash with Triton® X-100 permeabilization buffer (3 times, 2 minutes each)
  5. Add fluorescently labeled secondary antibody
  6. Incubate one hour at room temperature

Image

  1. Add PBS to each well
  2. Image cells with appropriate filters

 

Protocol tips

  • Leftover BrdU stock solution can be stored frozen for up to one year
  • Use short incubation time for rapidly proliferating cells, and longer incubation for slow-growing cells
  • Acid treatment separates DNA into single strands so the primary antibody can access the incorporated BrdU

NIH 3T3 cells showing incorporation of BrdU
NIH 3T3 cells showing incorporation of BrdU.