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P r o t o c o l s

Detection of BrdU Incorporation in DNA Synthesizing Cells

 


NOTE: Bromodeoxyuridine is a known carcinogen. Propidium iodine (PI) is known to be toxic and carcinogenic.

 

1.

 

Pulse actively growing cells in a tissue culture flask for one hour with 10 M BrdU (Sigma, Cat. No. B5002).

 

 

 

 

 

2.

 

Pour contents of tissue culture flask into a centrifuge tube. Centrifuge 10 minutes at 400 x g (all centrifugation steps) are performed at 400 x g, at RT). Aspirate supernatant. Loosen pellet by tapping tube.

 

 

 

 

 

3.

 

While vortexing, add ice cold 70% ethanol to cells, dropwise, to a final concentration of 1 x 10 6 cells/100 l. Incubate 20 minutes at RT.

 

 

 

 

 

4.

 

Aliquot 100 l into each test tube (12 mm x 75 mm). Wash with 1 ml wash buffer. Centrifuge 5 minutes. Aspirate supernatant. Loosen pellet.

 

 

 

 

 

5.

 

Resuspend pellet in denaturing solution. Mix well. Incubate 20 minutes at RT. NOTE: Denaturing solution must be made fresh.

 

 

 

 

 

6.

 

Add 1 ml wash buffer. Mix well. Centrifuge 5 minutes. Aspirate supernatant.

 

 

 

 

 

7.

 

Resuspend pellet in 0.5 ml 0.1 M sodium borate (Na 2 B 4 O 7 ), pH 8.5, to neutralize any residual acid. Incubate 2 minutes at RT.

 

 

 

 

 

8.

 

Add 1 ml wash buffer. Mix well. Centrifuge 5 minutes. Aspirate supernatant.

 

 

 

 

 

9.

 

Add primary antibody: dilute anti-BrdU monoclonal anitbody (Pharmingen Cat. No. 33281A) in dilution buffer, such that 50 l contains the optimal concentration. Resuspend cell pellet in 50 l of the diluted antibody. Incubate 20 minutes at RT.

 

 

 

 

 

10.

 

Add 1 ml wash buffer. Mix well. Centrifuge 5 minutes. Aspirate supernatant.

 

 

 

 

 

11.

 

Add secondary antibody: dilute FITC-conjugated goat anti-mouse Ig (PharMingen Cat. No. 12064D) in dilution buffer, such that 50 l contains the optimal concentration. Resuspend cell pellet in 50 l of the diluted antibody. Incubate 20 minutes at RT. NOTE: Eliminate this step when using FITC-conjugated anti-BrdU (Cat. No. 33284X)

 

 

 

 

 

12.

 

Add 1 ml wash buffer. Mix well. Centrifuge 5 minutes. Aspirate supernatant.

 

 

 

 

 

13.

 

Resuspend pellet in 0.5 ml propidium iodide (10 g/ml in PBS). Incubate 30 minutes at RT, protected from light.

 

 

 

 

 

14.

 

Analyze the cells by flow cytometry, exciting at 488 nm and measuring the BrdU-linked green fluorescence (FITC) through a 514 nm bandpass filter and the DNS linked red fluorescence (PI) through a 600 nm wave-length filter.

 

 

 

 

 

15.

 

Following analysis, flush flow cytometer for 10 minutes with 10% bleach and 5 minutes with dH 2 O.


SOLUTIONS:
Washing Solution: PBS containing 0.5% BSA
Denaturing Solution: 2M HCl
Dilution Buffer: PBS containing 0.5% Tween-20, 0.5% BSA

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Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis
Annexin V Staining Protocol
APO-BRDU™ Procedure
APO-DIRECT™ Procedure
Detection of BrdU Incorporation in DNA Synthesizing Cells
Staining Procedure for Flow Cytometric Detection of Human Cyclins
Indirect Immunofluorescence Staining of Human Platelets
Immunofluorescence Staining of Human Cells by Lysed Whole Blood Method
Immunofluorescent Staining of Mouse and Rat Leukocytes
The Uses of Fc Block in Immunophenotyping of Mouse or Rat Leukocytes
ELISA/ELISPOT
Cytokine ELISA
Mouse IgE ELISA Protocol
Immunohistochemistry
Preparation and Staining of Frozen Tissue Sections
Preparation and Staining of Paraffin Sections
Microwave citrate Pretreatment of Paraffin Sections
Immunohistochemistry / Tissue Section Staining
Cell Staining for Immunofluorescence Microscopy
Western Blotting
Western Blotting with Alkaline Phosphatase Conjugates
Western Blotting with Biotinylated Antibodies
Western Blotting with Horseradish Peroxidase Conjugates
Western Blotting with Monoclonal Antibodies
Western Blotting with Rabbit Polyclonal Antibodies
Preparation of Brain Membrane Fractions for Western Blot Analysis
Immunoprecipitation
Immunopurification of Tyrosine Phosphorylated Proteins
Immunoprecipitation with Antibody:Agarose Conjugates
Immunoprecipitation with anti-Phosphotyrosine: Biotin Conjugates
Immunoprecipitation With Soluble Antibodies
Immunoprecipitation of 35S Labeled Cells
RNAse Protection Assay (RPA)
RNase Protection Assay
Total RNA Isolation
Protein Kinase Assay (PKA)
Protein Kinase Assay with an Immunocomplex
Protein Kinase Assay after Denaturation and Renaturation




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