Encyclopedia of Experiments: Cancer Research
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Transcript
- 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 leukemia cells.
- Prior to antibody staining, treat the cells with DNAse. Resuspend the cells in 100 microliters of DNAse solution and incubate for one hour at 37 degrees Celsius. Add one milliliter of wash buffer, centrifuge the cells, and discard the supernatant. Antibody staining for intracellular markers other than BrdU can be performed simultaneously with the BrdU staining.
- It's important to prepare compensation controls consisting of unstained cells and cells labeled with each single fluorochrome. Ideally, use the same antibodies for compensation controls as those used in the experimental tubes.
- Resuspended the cells in 50 microliters of wash buffer with one microliter per 10 to the 6 cells of BrdU antibody. If using antibodies to other specific intracellular antigens, they should be added at this point. Incubate the cells for 20 minutes at room temperature.
After 20 minutes, wash the cells with one milliliter of wash buffer as shown earlier. Finally, stain the DNA for cell-cycle analysis. Loosen the pellet by flicking the tube and add 20 microliters of the 7-AAD solution. It is critical to use a constant amount of 7-AAD per cell. Resuspend the cells in one milliliter of staining buffer. Subsequently, perform flow cytometry analysis.
