Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

• US 4,780,406 A
• Filed: 06/05/1984
• Issued: 10/25/1988
• Est. Priority Date: 10/18/1983
• Status: Expired due to Term

• Current Assignee: University of California
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Gray, Joe W., Dolbeare, Frank A.
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): Wieder, Stephen C.
• Application Number:
  US06/617,655
• Time in Patent Office: 1,603 Days
• Field of Search: 435/6, 435/7, 435/29, 435/91, 436/63, 436/94, 436/56, 436/800, 935/76, 935/77
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/13 Tracers or tags
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method for the simultaneous flow cytometric measurement of the total DNA content and the level of DNA synthesis in normal and malignant cells is disclosed. The sensitivity of the method allows a study of cell cycle traverse rates for large scale cell populations as well as single cell measurements. A DNA stain such as propidium iodide or Hoechst 33258 is used as the probe for the measurement of total DNA content and a monoclonal antibody reactive with a DNA precursor such as halodeoxy-uridine (HdU), more specifically bromodeoxyuridine (BrdU) is used as a probe for the measurement of HdU or BrdU uptake by the cells as a measure of DNA synthesis.

Claims

1. A method for the simultaneous measurement of total cellular DNA content and of incorporated labeled halodeoxyuridine comprising:

providing halodeoxyuridine that is nonradioactively labeled;

reacting cells with the nonradioactively labeled halodeoxyuridine;

partially denaturing cellular DNA with a denaturing agent;

adding to the reaction medium monoclonal antibodies reactive with the nonradioactively labeled halodeoxyuridine;

reacting the bound monoclonal antibodies with a labeled second antibody;

incubating the mixture with a DNA stain; and

measuring simultaneously the intensity of the DNA stain and the amount of the bound labeled-antibody.

2. The method of claim 1 wherein said DNA stain is propidium iodide or Hoechst 33258.

3. The method of claim 1 wherein said halodeoxyuridine label is selected from the group consisting of an enzyme, fluorescent dye.

4. The method of claim 3 wherein said label is fluorescein or rhodamine.

5. The method of claim 1 wherein said second antibody is antimouse antibody.

6. The method of claim 5 wherein said antibody is labeled.

7. The method of claim 6 wherein said antibody label is an enzyme.

8. The method of claim of 7 wherein said enzyme is selected from the group consisting of alkaline phosphatase, beta-galactosidase and horse radish peroxidase.

9. The method of claim 6 wherein said antibody label is a fluorescent dye.

10. The method of claim 9 wherein said fluorescent dye is fluorescein or rhodamine.

11. The method of claim 1 wherein said halodeoxyuridine is bromodeoxyuridine, chlorodeoxyuridine or iododeoxyuridine.

12. The method of claim 11 wherein said halodeoxyuridine is bromodeoxyuridine.

13. A method for the bivariate analysis of cell cycle phase distribution of cellular DNA content comprising:

pulse labeling cells with labeled halodeoxyuridine;

denaturing the cellular DNA;

reacting said cells with monoclonal antibodies IU-1 or IU-2;

incubating the cells with a second antibody conjugated to an enzyme label or a fluorescent label;

incubating the cells with propidium iodide or Hoechst 33258;

measuring simultaneously the bound halodeoxyuridine label and the bound propidium iodide or Hoechst 33258 cytometrically or spectrophotometrically as a measure of the incorporated HdU and of the total DNA in said cells; and

analyzing the bivariate distribution of total DNA and bound bromodeoxyuridine through the various phases of the cell cycle.

14. The method of claim 13 wherein said halodeoxyuridine is bromodeoxyuridine, chlorodeoxyuridine or iododeoxyuridine.

15. A method for the bivariate analysis of cell cycle traverse comprising:

pulse labeling cells with halodeoxyuridine;

reacting said cells with mabs IU-1 or IU-2;

incubating the cells with a second antibody conjugated to an enzyme label or a fluorescent label;

incubating the cells with a DNA stain;

measuring simultaneously at specified time intervals the bound halodeoxyuridine label and the bound DNA stain cytometrically or spectrophotometrically as a measure of the incorporated halodeoxyuridine and of the total DNA in said cells; and

analyzing the bivariate distribution of total DNA and bound bromodeoxyuridine through the various phases of the cell cycle.

16. The method of claim 15 wherein said halodeoxyuridine is bromodeoxyuridine, chlorodeoxyuridine, or iododeoxyuridine.

17. The method of claim 15 wherein said DNA stain is propidium iodide or Hoechst 33258.

18. A method for the measurement of halodeoxyuridine incorporation following drug treatment of an animal comprising:

treating said animal with a drug;

administering halodeoxyuridine to said drug-treated animal;

selectively removing cells from the body of said animal;

reacting said cells and monoclonal antibodies reactive with halodeoxyuridine;

incubating said cells with a second antibody conjugated to a label;

incubating said cells with a DNA stain;

measuring simultaneously at specified time invervals the bound halodeoxyuridine and the bound DNA stain cytometrically or spectrophotometrically as a measure of the incorporated halodeoxyuridine and of the total DNA in said cells; and

analyzing the bivariate distribution of total DNA and bound bromodeoxyuridine through the various phases of the cell cycle.

19. The method of claim 18 wherein said halodeoxyuridine is labeled.

20. The method of claim 19 wherein said label is a radionuclide.

21. The method of claim 20 wherein said halodeoxyuridine is bromodeoxyuridine or iododeoxyuridine.

22. The method of claim 18 wherein said second antibody label is a fluorescent dye or an enzyme.

23. The method of claim 22 wherein said dye is fluorescein or rhodamine.