NIH 3T3 cells showing incorporation of BrdU.
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)
- Invitrogen Anti-BrdU monoclonal antibody (such as Cat. No. B35130)
- Invitrogen 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% Invitrogen 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
- Dissolve 100 mg BrdU in 32.5 mL anhydrous DMSO (10 mM stock solution)
- 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
- Culture cells in appropriate vessel for microscopy
- Remove culture medium from cells and replace with BrdU labeling solution
- Incubate cells at 37°C for 2 hours
- Remove labeling solution and wash two times with PBS
- Wash with PBS (3 times, 2 minutes each)
Fix, permeabilize, and acid-wash
- Remove PBS and add 1 mL of 3.7% formaldehyde in PBS to each well
- Incubate for 15 minutes at room temperature
- Wash with PBS (3 times, 2 minutes each)
- Remove PBS and add 1 mL of Triton X-100 permeabilization buffer to each well
- Incubate for 20 minutes at room temperature
- Remove permeabilization buffer and add 1 mL of 1N HCl
- Incubate 10 minutes on ice
- Remove this solution and add 1 mL of 2N HCl
- Incubate 10 minutes at room temperature
- Add 1 mL phosphate/citric acid buffer, pH 7.4
- Incubate 10 minutes at room temperature
- Wash with Triton X-100 permeabilization buffer (3 times, 2 minutes each)
Detect incorporated BrdU
- Remove this solution and add 1 mL of antibody staining buffer
- Add anti-BrdU primary antibody
- Incubate overnight at room temperature
- Wash with Triton X-100 permeabilization buffer (3 times, 2 minutes each)
- Add fluorescently labeled secondary antibody
- Incubate one hour at room temperature
Image
- Add PBS to each well
- 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