Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

• US 4,585,736 A
• Filed: 10/18/1983
• Issued: 04/29/1986
• Est. Priority Date: 10/18/1983
• Status: Expired due to Term

• Current Assignee: University of California
• Original Assignee: United States Department of Energy (Government of the United States of America)
• Inventors: Gray, Joe W., Dolbeare, Frank A.
• Primary Examiner(s): Nucker, Christine M.
• Application Number:
  US06/542,955
• Time in Patent Office: 924 Days
• Field of Search: 436/508, 436/517, 436/518, 436/63, 436/94, 435/4, 435/6, 435/7, 435/29, 435/91, 435/172, 435/3, 935/77, 935/108
• 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: 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 is used as the probe for the measurement of total DNA content and a monoclonal antibody reactive with a DNA precursor such as bromodeoxyuridine (BrdU) is used as a probe for the measurement of 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 halodeoxyuridine comprising:

reacting cells with radioactive labeled halodeoxyuridine;

denaturing cellular DNA with a denaturing agent;

adding to the reaction medium monoclonal antibodies reactive with 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 as a measure of the total cellular DNA and the halodeoxyuridine incorporated as a measure of DNA synthesis.

2. The method of claim 1 wherein said radioactive label is tritium.

3. The method of claim 2 wherein said DNA stain is propidium iodide.

4. The method of claim 3 wherein said second antibody label is an enzyme.

5. The method of claim 4 wherein said enzyme is selected from the group consisting of alkaline phosphatase, beta-galactosidase and peroxidase.

6. The method of claim 3 wherein said antibody label is a fluorescent dye.

7. The method of claim 6 wherein said fluorescent dye is fluorescein or rhodamine.

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

9. The method of claim 8 wherein said HdU is BrdU.

18. The method of claim 1 wherein said denaturing agent is HCl.

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

labeling cells with radioactive nuclide labeled halodeoxyuridine;

denaturing the cellular DNA;

reacting said cells with monoclonal antibodies produced by hybridoma identified as HB-8320 or HB-8321;

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

incubating the cells with propidium iodide;

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

11. The method of claim 10 wherein said halodeoxyuridine bromodeoxyuridine, chlorodeoxyuridine, fluorodeoxyuridine or iododeoxyuridine.

19. The method of claim 10 wherein said cellular DNA is denatured with HCl.

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

labeling cells with radioactively labeled halodeoxyuridine;

denaturing the cellular DNA with a denaturing agent;

reacting said cells with monoclonal antibodies produced by hybridoma identified as HB-8320 or HB-8321;

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

incubating the cells with propidium iodide;

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

measuring the bivariate distribution of total DNA and bound halodeoxyuridine through the various phases of the cell cycle.

13. The method of claim 12 wherein said HdU is bromodeoxyuridine, chlorodeoxyuridine, fluorodeoxyuridine or iododeoxyuridine.

14. A method for the measurement of DNA synthesis following drug treatment comprising:

treating mice with araC;

administering to said araC-treated mice halodeoxyuridine;

reacting said cells with monoclonal antibodies produced by hybridoma identified as HB-8320 or HB-8321;

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

incubating the cells with propidium iodide;

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

measuring the bivariate distribution of total DNA and bound halodeoxyuridine through the various phases of the cell cycle.

15. The method of claim 14 wherein said HdU is BrdU, CldU or IdU.

16. The method of claim 15 wherein said second antibody label is a fluorescent dye.

17. The method of claim 16 wherein said dye is fluorescein.

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

labeling cells with radioactively labeled halodeoxyuridine;

denaturing the cellular DNA;

reacting said cells with monoclonal antibodies produced by hybridoma identified as HB-8320 or HB-8321;

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

incubating the cells with a DNA stain;

measuring simultaneously 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

measuring the bivariate distribution of total DNA and bound halodeoxyuridine through the various phases of the cell cycle.

21. The method of claim 20 wherein said denaturing agent is HCl and said DNA stain is propidium iodide.

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

labeling cells with tritiated halodeoxyuridine;

denaturing the cellular DNA with a denaturing agent;

reacting said cells with monoclonal antibodies produced by hybridoma identified as HB-8320 or HB-8321;

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

incubating the cells with a suitable 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

measuring the bivariate distribution of total DNA and bound halodeoxyuridine through the various phases of the cell cycle.

23. The method of claim 22 wherein said denaturing agent is HCl and said DNA stain is propidium iodide.