Flow cytometry antibodies, kits, reagents

BrdU staining of cells for cell cycle analysis and apoptosis

BrdU Staining of Cells for Cell Cycle Analysis and Apoptosis

BrdU Staining of Cells for Cell Cycle Analysis and Apoptosis for Flow Cytometry

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BrdU is an analogue of thymidine readily incorporated into DNA during DNA synthesis and is an accurate method to monitor proliferation and apoptosis. The anti-BrdU antibody mouse monoclonal MCA2483 (clone Bu20a) and rat monoclonal MCA2060 (clone BU1/75 (ICR1)) are suitable for flow cytometry. The following methods were used and provide a useful guide for using anti-BrdU antibodies.


Phosphate buffered saline (PBS) (BUF036A) containing 1% bovine serum albumin (PBS/BSA)
2 M HCl containing 0.5% Triton X-100
0.05% (v/v) Tween -20 in PBS
Propidium iodide
0.1 M Na2B4O7, pH 8.5


  1. Add BrdU to the cell suspension in culture medium to a final concentration of 10 μM and incubate for at least 30 min at 37°C in a CO2 incubator.
  2. Wash cells twice with PBS/BSA, at 500 x g for 10 min at room temperature, decant supernatant.
  3. Resuspend in 2-5 ml cold (4oC) 70% ethanol. Add drop wise to cell pellet while vortexing. Fix for at least 30 min on ice.
  4. Centrifuge at 500 x g for 10 minutes, decant supernatant.
  5. Resuspend the pellet in 2 ml of 2 N HCl containing 0.5% Triton X-100. Incubate for 30 min at room temperature (preferably on a rocking platform).
  6. Centrifuge at 500 x g for 10 minutes, decant supernatant. Resuspend in 3 ml of 0.1 M Na2B4O7, pH 8.5 for 2 min at room temperature.
  7. Centrifuge at 500 x g for 10 min, decant supernatant, and resuspend in room temperature PBS/BSA + 0.05% Tween-20. Adjust cell concentration to 1 × 107 cells/ml.
  8. Aliquot 100 μl of the cell suspension into required number of FACS tubes.
  9. Incubate with antibody at the recommended vendor dilution overnight at 4oC avoiding direct light.
  10. Resuspend in 2 ml of room temperature PBS/BSA. Centrifuge at 500 x g for 10 min at room temperature.
  11. If a secondary antibody is required, then decant the supernatant, add 100 ul of PBS/BSA and incubate with the secondary antibody at the vendor recommended dilution for at least 30 min at 4oC.
  12. Wash with 2 ml of PBS/BSA, centrifuge at 500 x g for 10 min.
  13. Re-suspend cells in 1 ml of PBS. Add propidium iodide e.g. 1-2 drops of ReadiDrop™ propidium iodide (135-1101).
  14. Analyze by flow cytometry. The propidium iodide should be read on the appropriate channel in the linear scale. Doublets should be gated out using the Area vs Height or Width depending on your instrument.


The acid treatment to unwind the DNA may affect surface immunophenotyping. Staining of cells with BrdU using DNAseI may be applicable if this is required.

Appropriate controls should always be carried out, for flow cytometry the following should be considered for inclusion;

  • A known positive sample
  • Isotype controls used to determine if the staining is specific
  • Unstained cells should always be included in the experimental set-up to monitor autofluorescence

For all multi-color flow cytometry experiments it is advisable to include compensation controls and Fluorescence Minus One (FMO) controls, which assist with identifying gating boundaries.