BrdU Staining Protocol

Principles of BrdU Staining

BrdU staining is based on the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of dividing cells. BrdU is a derivative of uridine that, when added to cultured cells, incorporates substituted thymidine into the DNA of cells in the S phase. During the staining process, the DNA must be degraded by DNase I, which is critical to the staining success. Staining was then performed with an anti-BrdU antibody to identify the incorporation of BrdU into proliferating S-phase cells. BrdU staining is an advantageous alternative for customers assessing cell proliferation using traditional [3H]thymidine incorporation methods. BrdU staining can help customers identify cells that have entered the S-proliferative phase triggered by stimulators or proliferation-inducing treatments.

BrdU Labeling

In Vitro BrdU Labeling

1) Dissolve 3 mg of BrdU in 1 mL of water to prepare a 10 mM BrdU stock solution.
2) Dilute 10 mM BrdU stock solution in cell culture medium to prepare 10 μM BrdU labeling solution.
3) Filter 10 µM BrdU labeling solution through a 0.2 µm filter (sterile conditions).
4) Replace existing medium in cells with 10 μM labeling solution.
5) Incubate cells in the BrdU labeling solution in a CO₂ incubator at 37ºC for 1-24 hours.
Note: BrdU incubation time depends on cell division rate. The exact time needed to achieve the best signal-to-noise ratio should be optimized.
6) Remove the BrdU labeling solution from cells.
7) Wash twice in PBS for approximately 5 seconds each.
8) Wash three times in PBS for two minutes each.
9) Fix and permeabilize cells according to standard immunocytochemistry (ICC)/immunofluorescence (IF) protocols. See below for DNA hydrolysis steps prior to immunostaining.

In Vivo BrdU Labeling

Oral administration and intraperitoneal injection are common methods of labeling cells with BrdU in vivo.

DNA Hydrolysis

Co-staining with Anti-BrdU Antibodies

In addition to BrdU staining, co-staining with other antibodies (e.g., Ki67, NeuN, Doublecortin) can provide valuable information about the localization and function of proliferating cells. For example, NeuN, which is a marker of mature neurons, can be used in conjunction with BrdU staining to identify newly differentiated neurons.