BrdU Immunofluorescence Staining: A Technique to Identify Cells in Different Phases of Cell Cycle

- Bromodeoxyuridine or BrdU is a synthetic thymidine nucleoside analog. During the synthesis phase of the cell cycle, BrdU substitutes thymidine and integrates into the newly synthesized DNA. The incorporated BrdU allows the tracking of cell-cycle progression. To detect the incorporated BrdU, first, fix and permeabilize the cells to render them permeable.

Next, treat the cells with DNAse enzyme to cleave the DNA strands and expose the incorporated BrdU. Subsequently, add a suitable fluorescent labeled anti-BrdU antibody. The antibody enters the nucleus and readily binds to the exposed BrdU present in the DNA.

Additionally, treat the cells with a suitable fluorescent DNA-binding dye. The dye binds to the DNA, allowing the quantification of total DNA content. Use flow cytometry to detect the fluorescence from both labeled BrdU and DNA.

Fluorescence intensities from the labeled DNA differentiate cells in different cell-cycle phases based on the variation in total DNA content. The fluorescent signal from BrdU identifies the current cell cycle position of BrdU-incorporated cells.

In this example, we will perform the BrdU immunofluorescent staining of NALM-6-- acute lymphoblastic leukemi