Protocol: BrdU Staining of Cells for Cell Cycle Analysis

Reagents

• Cell culture medium
• Phosphate buffered saline, 10x (PBS) (BUF036A)
• PBS containing 1% bovine serum albumin (PBS/BSA)
• BrdU (Merck, 19-160)
• 70% ethanol in distilled water
• 2 M hydrochloric acid (HCl) containing 0.5% octylphenol ethoxylate (Triton X-100; Merck, TX1568)
• 0.05% (v/v) Tween 20 in PBS
• 0.1 M Na_²B_⁴O_⁷, pH 8.5
• Anti-BrdU Antibody (MCA2483 or AHP2405)
• ReadiDrop Propidium Iodide (1351101)

Methods

1. Treat suspension cells with BrdU in cell culture medium to a final concentration of 10 μM. Incubate for at least 30 min at 37°C in a CO_² incubator.
2. Transfer cells to a 50 mL centrifuge tube and add 10 mL PBS/BSA. Centrifuge at 500 x g for 10 min at room temperature (RT) and discard supernatant.
3. Repeat step 2.
4. Resuspend cells in 2–5 mL cold (4°C) 70% ethanol. Add dropwise to cell pellet while vortexing. This should ensure fixation of all cells and minimize clumping.
5. Incubate for at least 30 min on ice.
6. Centrifuge at 500 x g for 10 min and discard supernatant.
7. Resuspend cells in 2 mL of 2 M HCl containing 0.5% octylphenol ethoxylate (Triton X-100; Merck TX1568). Incubate for 30 min at RT, preferably on a rocking platform set to 15 rpm.
8. Centrifuge at 500 x g for 10 min and discard supernatant.
9. Resuspend cells in 3 mL of 0.1 M Na_²B_⁴O_⁷, pH 8.5 and incubate for 2 min at RT.
10. Centrifuge at 500 x g for 10 min, discard supernatant, and resuspend in RT PBS/BSA + 0.05% Tween 20. Adjust cell concentration to 1 × 10⁷ cells/mL.
11. Aliquot 100 μL of the cell suspension into required number of 5 mL tubes.
12. Incubate with Anti-BrdU Antibody (MCA2483 or AHP2405) at the dilution determined from a titration experiment or the vendor-recommended dilution. Mix well and incubate at 4°C overnight, avoiding direct light.
13. Add 2 mL of RT PBS/BSA. Centrifuge at 500 x g for 10 min at RT and discard supernatant.
14. Optional: When using an unconjugated antibody, perform incubation with a secondary antibody. Resuspend cells in 100 μL of PBS/BSA and incubate with the secondary antibody at a dilution determined from a titration experiment or the vendor-recommended dilution for at least 45 min at RT. Wash with 2 mL of PBS/BSA, centrifuge at 500 x g for 10 min, and discard supernatant.
15. Resuspend cells in 1 mL of PBS.
16. Add 1–2 drops of ReadiDrop Propidium Iodide (1351101) and vortex briefly.
17. Acquire samples on a flow cytometer.

Notes

• The optimal BrdU incubation time is dependent on culture conditions and the proliferation profile of your cells and therefore should be optimized for your experiment
• The acid treatment to unwind the DNA may affect surface immunophenotyping. If surface staining is required, using BrdU with DNAse I may be required
• When acquiring cells, select the appropriate laser and filter for propidium iodide and collect data in the linear scale
• Suitable alternative DNA binding dyes to propidium iodide include 7-aminoactinomycin-D (1351102) and Hoeschst 33342 (1351304)
• All antibodies should be titrated prior to use to ensure the optimal concentration is used