Bivariate Cell Cycle Assay (BrdU/PI)
Studies have shown that cancer is a cell cycle-related disease cause by the loss of cell cycle regulation. Therefore, cell cycle analysis plays an increasingly important role in cancer research, anticancer drug screening, mechanism clarification, and cytokinetic research. The traditional single parameter DNA content analysis can only identify G0/G1 phase, S phase, and G2/M phase, but cannot distinguish G0 from G1 phase or G2 from M phase. Bivariate cell cycle assay using cell cycle-specific antibodies and DNA dyes will not only reveals the distribution of cells at particular cycle phases, but also allows researchers to elucidate the molecular and functional mechanisms within the cell cycle. This method is very sensitive and can be used to screen in vivo inhibitors involved in the S-phase transition by flow cytometry.
Materials
DNase I
Wash buffer
Assay buffer
Cell growth medium
Permeabilization buffer
Propidium iodide/RNase solution
Anti-BrdU-FITC antibody
Fixation buffer
Flow cytometry
Centrifuge
Labeling of Cells with BrdU
1) For adherent cells, culture cells in growth media until 70-80% confluent prior to cell seeding for assay.
2) For suspension cells, the cell density should not exceed 2 x 106 cells/mL.
3) Incubate the cells with 10-50 μM BrdU for 60 minutes in the dark.
Note: Different BrdU concentrations and different incubation times may be optimized depending on the cell type and experimental purpose/design.
BrdU and PI Staining Procedures
This assay protocol is applicable to proliferation/cell cycle analysis of a variety of human cell types.
- Harvest cells
1) For adherent cells, cell culture is obtained by using a mild enzyme such as trypsin or EDTA-based cell dissociation buffer.
2) For suspension cells, cell culture is obtained by gently pipetting cell suspension up and down to ensure complete homogeneity.
3) Remove cell culture media by centrifugation at 300 g for 5 minutes.
Note: It is recommended to prepare two more samples for single color staining. One sample for BrdU staining only and the other sample for PI staining only. These samples will be used for compensation adjustment of test samples. - Fix and permeabilize cells
1) For every 1 x 106 cells, resuspend with 100 µL of wash buffer and add 100 µL of fixation buffer. Incubate for 20 minutes on ice.
2) Remove fixation buffer by centrifugation at 600 g for 5 minutes. Aspirate and discard the supernatant, only saving the cell pellet.
3) For every 1 x 106 cells, resuspend with 100 µL ice cold permeabilization buffer. Allow to incubate for 5 minutes on ice.
4) Remove permeabilization buffer by centrifugation at 600 g for 5 minutes. Aspirate and discard the supernatant, only saving the cell pellet.
5) Wash cells once by adding 200 µL assay buffer for every 1 x 106 cells. Carefully disrupt cell pellet to homogeneity, and remove assay buffer by centrifugation at 600 g for 5 minutes. Aspirate and discard the supernatant, only saving the cell pellet. - DNA denaturation
1) Add 100 µL of diluted DNase I (diluted to 300 µg/mL in assay buffer) to each sample and incubate for 1 hour at 37°C.
2) Remove DNA denaturation buffer by centrifugation at 600 g for 5 minutes. Aspirate and discard the supernatant, only saving the cell pellet.
3) Wash cells once by adding 200 µL assay buffer for every 1 x 106 cells. Again, carefully disrupt cell pellet to homogeneity and remove assay buffer by centrifugation at 600 g for 5 minutes. Aspirate and discard the supernatant, only saving the cell pellet. - BrdU staining
1) Add 100 µL of Anti-BrdU-FITC antibody for every 1 x 106 cells. Allow to incubate on ice for 1 hour in the dark.
2) Wash cells by adding 200 µL of assay buffer for every 1 x 106 cells. Carefully disrupt cell pellet to homogeneity and remove assay buffer by centrifugation at 600 g for 5 minutes. Aspirate and discard the supernatant, only saving the cell pellet.
3) Perform washing step twice to remove any excess Anti-BrdU-FITC antibody. - DNA staining
1) Add 200 µL of freshly prepared propidium iodide/RNase solution. Incubate for 30 minutes at room temperature in the dark. - Flow cytometry analysis
1) Measure the BrdU-associated green fluorescence and DNA-associated red fluorescence.
2) DNA measurement must be analyzed using linear scaling. Analysis using logarithmic scale will make it difficult to discern cellular distributions at each phase of the cell cycle.
Troubleshooting
| Potential problems | Experimental suggestions |
| Loss or lack of signal | A substantial decrease in cell numbers can lead to a loss of signal. Make sure the cell density remains at approximately 0.5 x 105 cells/mL during analysis. |
| Lack of signal may indicate that more antibodies are needed. Further antibody titrations may be necessary for some cell types to capture the ideal staining effect. | |
| Non-specific staining | Non-specific staining and background may indicate that less antibody is required, so further antibody titrations may be necessary for some cell types. |
| Poor repeatability | Monitor experimental cells to ensure the cell viability and cell numbers being analyzed are consistent. Any drop in cell numbers or viability will affect the experimental results. |
References
- Juan G, et al.; Methods to identify mitotic cells by flow cytometry. Meth Cell Biol, 2001, 63: 343-354.
- Sherr C. J. The pezcoller lecture: cancer cell cycles revisited. Cancer Res, 2000, 60: 3689-3695.
- Nunez R. DNA measurement and cell cycle analysis by flow cytometry. Curr Issues Mol Biol, 2001, 3(3): 67-70.
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