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Immunofluorescence Assay for S Phase Entry Using BrdU Incorporation
Published: Feb 5, 2012 DOI: 10.21769/BioProtoc.197 Views: 19363
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Abstract
Bromodeoxyuridine (BrdU) is thymidine analogue and can incorporated into the newly synthezised DNA of S-phase cells, the antibody specific for BrdU can then be used to detect the incorporated BrdU, thus indicating cells that were actively replicating their DNA and estimating for the percentage of cells in S-phase. The immunocytochemical detection of BrdU incorporated into DNA is a powerful tool to study the cytokinetics of normal and tumor cells. In vitro or in vivo labeling of tumor cells with BrdU and the subsequent detection of incorporated BrdU with specific anti-BrdU monoclonal antibodies is an accurate and comprehensive method to quantitate the degree of DNA-synthesis.
Keywords: Bromodeoxyuridine(BrdU)
S-phase
Immunofluorescence
Materials and Reagents
- BrdU (Sigma-Aldrich, catalog number: B5002 )
- Phosphate buffered saline (PBS)
- Methanol (Thermo Fisher Scientific, catalog number: BP1105-4 )
- BSA (Sigma-Aldrich, catalog number: A3803 )
- HCl
- NaN3
- Triton X-100
- Monoclonal anti-BrdU (Sigma-Aldrich, catalog number: B2531 )
- Alexa 488-conjugated goat anti-mouse immunoglobulin G antibody (Life Technologies, Molecular Probes®/Alexa Fluor® 488, catalog number: A-11008 or A-11034 )
- DAPI (make 1 mg ml-1 stock, 1,000x) (Boeringer Manheim 236 276 )
- Coverglasses (12 mm) (Thermo Fisher Scientific, catalog number: 12-545-82 )
- Fluormount-GTM Mounting solution (SouthernBiotech, catalog number: 0100-01 )
- PBS-BT solution (see Recipes)
Equipment
- Centrifuges (Beckman Falcon, model: TLS-55 )
- Parafilm
- 6-well plate
- 24-well plate
Procedure
Category
Cancer Biology > General technique > Cell biology assays
Cancer Biology > Proliferative signaling > Cell biology assays
Cell Biology > Cell imaging > Fluorescence
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