US4585736A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Flow cytometric measurement of total DNA and incorporated halodeoxyuridine
Assignee: United States, ENERGY THE, Secretary of, Department of
•
Filing: 1983-10-18
•
Granted: 1986-04-29
•
Expires: 2003-10-18
Abstract
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.
Inventors
- 👤 Frank A. Dolbeare
- 👤 Joe W. Gray
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 19:31
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/5308; C; C12; C12Q; C12Q1/00; C12Q1/68; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
83 words
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.
Claim 2
Independent Claim
83 words
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.
Claim 3
Dependent Claim
11 words
The method of claim 1 wherein said radioactive label is tritium.
Claim 4
Dependent Claim
12 words
The method of claim 2 wherein said DNA stain is propidium iodide.
Claim 5
Dependent Claim
13 words
The method of claim 3 wherein said second antibody label is an enzyme.
Claim 6
Dependent Claim
20 words
The method of claim 4 wherein said enzyme is selected from the group consisting of alkaline phosphatase, beta-galactosidase and peroxidase.
Claim 7
Dependent Claim
13 words
The method of claim 3 wherein said antibody label is a fluorescent dye.
Claim 8
Dependent Claim
13 words
The method of claim 6 wherein said fluorescent dye is fluorescein or rhodamine.
Claim 9
Dependent Claim
14 words
The method of claim 1 wherein said halodeoxyuridine is bromodeoxyuridine, chlorodeoxyuridine, fluorodeoxyuridine or iododeoxyuridine.
Claim 10
Dependent Claim
10 words
The method of claim 8 wherein said HdU is BrdU.
Claim 11
Independent Claim
87 words
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.
Claim 12
Independent Claim
87 words
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.
Claim 13
Dependent Claim
13 words
The method of claim 10 wherein said halodeoxyuridine bromodeoxyuridine, chlorodeoxyuridine, fluorodeoxyuridine or iododeoxyuridine.
Claim 14
Independent Claim
108 words
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.
Claim 15
Independent Claim
108 words
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.
Claim 16
Dependent Claim
14 words
The method of claim 12 wherein said HdU is bromodeoxyuridine, chlorodeoxyuridine, fluorodeoxyuridine or iododeoxyuridine.
Claim 17
Independent Claim
105 words
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.
Claim 18
Independent Claim
105 words
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.
Claim 19
Dependent Claim
13 words
The method of claim 14 wherein said HdU is BrdU, CldU or IdU.
Claim 20
Dependent Claim
14 words
The method of claim 15 wherein said second antibody label is a fluorescent dye.
Claim 21
Dependent Claim
10 words
The method of claim 16 wherein said dye is fluorescein.
Claim 22
Dependent Claim
11 words
The method of claim 1 wherein said denaturing agent is HCl.
Claim 23
Dependent Claim
13 words
The method of claim 10 wherein said cellular DNA is denatured with HCl.
Claim 24
Independent Claim
106 words
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.
Claim 25
Independent Claim
106 words
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.
Claim 26
Dependent Claim
18 words
The method of claim 20 wherein said denaturing agent is HCl and said DNA stain is propidium iodide.
Claim 27
Independent Claim
109 words
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.
Claim 28
Independent Claim
109 words
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.
Claim 29
Dependent Claim
18 words
The method of claim 22 wherein said denaturing agent is HCl and said DNA stain is propidium iodide.
References Cited (Backward Citations)
US4196265A
Cited
EP0102228A2
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1989-04-28 | 1989-10-28 | 1990-04-29 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-04-28 | 1993-10-28 | 1994-04-29 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-04-28 | 1997-10-28 | 1998-04-29 | $7700.00 | Window Closed / Lapsed |