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US4906561A
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Nuclear isolation medium and procedure for separating cell nuclei
Assignee: Individual
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Filing: 1987-04-06
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Granted: 1990-03-06
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Expires: 2007-04-06
Abstract
Abstract Nuclear isolation media and procedures are described for dissociating discrete, non-agglomerated cell nuclei from animal tissue without the need to use enzyme treatments, centrifugation or the like in order to achieve the desired separation. The media facilitates separation and maintains the nuclear membrane intact and in its normal physiological environment. When a DNA-fluorochrome stain is included in the medium an essentially one step combination nuclear isolation and DNA staining procedure is used to measure DNA in tissue cells by flow cytometry. Rapid and consistant results are obtained and multiple sampling of the same tissue or comparison between whole tissues and their single cell isolates are also described.
Inventors
- 👤 Jerry T. Thornthwaite
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 14:40
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/5005; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6806; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10T; Y10T436/00; Y10T436/10; Y10T436/107497; Y10T436/108331; Y10T436/25; Y10T436/25375
Claims Analysis
Total Claims: 41
Claim 1
Independent Claim
93 words
A nuclear isolation medium for dissociating a tissue or cell sample into discrete, non-agglomerated nuclei, the medium comprising a solution of a nonionic surfactant present in an amount sufficient to separate the cell nuclei while maintaining nuclear integrity dissolved in an aqueous solution that is substantially or completely isotonic with and maintains the normal electronic volume of the cell nuclei, the nuclear isolation medium further including divalent cations in an amount substantially equal to the amounts of said cations in the fluid surrounding the nuclear membrane environment of the tissue or cell sample.
Claim 2
Dependent Claim
93 words
A nuclear isolation medium for dissociating a tissue or cell sample into discrete, non-agglomerated nuclei, the medium comprising a solution of a nonionic surfactant present in an amount sufficient to separate the cell nuclei while maintaining nuclear integrity dissolved in an aqueous solution that is substantially or completely isotonic with and maintains the normal electronic volume of the cell nuclei, the nuclear isolation medium further including divalent cations in an amount substantially equal to the amounts of said cations in the fluid surrounding the nuclear membrane environment of the tissue or cell sample.
Claim 3
Dependent Claim
15 words
The nuclear isolation medium of claim 1 wherein magnesium and/or calcium are the divalent cations.
Claim 4
Dependent Claim
25 words
The nuclear isolation medium of claim 1 further including serum albumin in an amount substantially the same as the physiological state of the tissue sample.
Claim 5
Dependent Claim
26 words
The nuclear isolation medium of claim 3 wherein the divalent cations are magnesium and/or calcium and the serum albumin concentration is about 0.05% to 1.0% v/v.
Claim 6
Dependent Claim
12 words
The nuclear isolation medium of claim 1, further including a microscopy stain.
Claim 7
Dependent Claim
100 words
A non-enzymatic method of dissociating cell nuclei from tissue containing same into discrete, non-agglomerated individual cell nuclei, while maintaining nuclear integrity said method comprising the steps of: (a) placing a tissue sample in a nuclear isolation medium containing an isotonic solution of a cell-separating amount of a nonionic surfactant; (b) physically dissociating the tissue sample while in contact with the nuclear isolation medium; (c) allowing the dissociated tissue sample to remain in contact with the nuclear isolation medium until a quantity of single nuclei have been separated; and (d) removing the single nuclei from the remaining tissue and cellular debris.
Claim 8
Dependent Claim
100 words
A non-enzymatic method of dissociating cell nuclei from tissue containing same into discrete, non-agglomerated individual cell nuclei, while maintaining nuclear integrity said method comprising the steps of: (a) placing a tissue sample in a nuclear isolation medium containing an isotonic solution of a cell-separating amount of a nonionic surfactant; (b) physically dissociating the tissue sample while in contact with the nuclear isolation medium; (c) allowing the dissociated tissue sample to remain in contact with the nuclear isolation medium until a quantity of single nuclei have been separated; and (d) removing the single nuclei from the remaining tissue and cellular debris.
Claim 9
Dependent Claim
15 words
The cell nuclei dissociation method of claim 6 wherein the nuclear isolation medium is buffered.
Claim 10
Dependent Claim
19 words
The cell nuclei dissociation method of claim 6 wherein the nuclear isolation medium also contains at least one stain.
Claim 11
Dependent Claim
36 words
The cell nuclei dissociation method of claim 6 wherein the nuclear isolation medium also contains serum albumin in an amount substantially the same as the physiological state of the animal from which the tissue was taken.
Claim 12
Dependent Claim
26 words
The cell nuclei dissociation method of claim 9 wherein the serum albumin is present in an amount of from about 0.05% to about 1.0% by weight.
Claim 13
Dependent Claim
39 words
The cell nuclei dissociation method of claim 6 wherein the nuclear isolation medium also contains divalent cations in an amount substantially equal to the amounts of said cations in the fluid surrounding the nuclear membrane of the tissue sample.
Claim 14
Dependent Claim
18 words
The cell nuclei dissociation method of claim 11 wherein the divalent cations are calcium, magnesium or their mixtures.
Claim 15
Dependent Claim
62 words
The cell nuclei dissociation method of claim 11 wherein the nuclear isolation medium also contains: serum albumin in an amount substantially the same as the physiological state of the animal from which the tissue was taken, and magnesium plus calcium ions in amounts substantially equal to the amounts of said cations in the fluid surrounding the nuclear membrane of the tissue sample.
Claim 16
Dependent Claim
26 words
The cell nuclei dissociation method of claim 6 wherein the single cell nuclei are removed from the tissue and cellular debris in step (d) by filtration.
Claim 17
Dependent Claim
21 words
The cell nuclei dissociation method of claim 6 including the additional step of: (e) subjecting the separated nuclei to cytometric analysis.
Claim 18
Dependent Claim
15 words
The method of claim 6 further comprising the step of calculating the electronic nuclear volume.
Claim 19
Dependent Claim
124 words
A non-enzymatic method of preparing tissue for cytometric analysis by dissociating cell nuclei from an animal tissue containing same into discrete, non-agglomerated individual cell nuclei, while maintaining nuclear integrity said method comprising the steps of: (a) placing a tissue sample in a nuclear isolation medium containing a solution of a cell-separating amount of a nonionic sursatant and a DNA stain; (b) physically dissociating the tissue sample while in contact with the nuclear isolation medium; (c) allowing the dissociated tissue sample to remain in contact with the nuclear isolation medium until at least an analyzable quantity of single nuclei have been separated; (d) filtering and removing the single nuclei from the tissue and cellular debris, and (e) subjecting the thus separated nuclei to cytometric analysis.
Claim 20
Dependent Claim
124 words
A non-enzymatic method of preparing tissue for cytometric analysis by dissociating cell nuclei from an animal tissue containing same into discrete, non-agglomerated individual cell nuclei, while maintaining nuclear integrity said method comprising the steps of: (a) placing a tissue sample in a nuclear isolation medium containing a solution of a cell-separating amount of a nonionic sursatant and a DNA stain; (b) physically dissociating the tissue sample while in contact with the nuclear isolation medium; (c) allowing the dissociated tissue sample to remain in contact with the nuclear isolation medium until at least an analyzable quantity of single nuclei have been separated; (d) filtering and removing the single nuclei from the tissue and cellular debris, and (e) subjecting the thus separated nuclei to cytometric analysis.
Claim 21
Dependent Claim
33 words
The method of claim 17 wherein the nuclear isolation medium also contains serum albumin in an amount substantially the same as the physiological state of the animal from which the tissue was taken.
Claim 22
Dependent Claim
38 words
The method of claim 18 wherein the nuclear isolation medium additionally contains magnesium and/or calcium ions in an amount substantially equal to the amounts of said ions in the fluid surrounding the nuclear membrane of the tissue sample.
Claim 23
Independent Claim
148 words
The method of comparing normal cell nuclei with suspected cell nuclei both said nuclei of the same histological type, said method comprising the steps of: (1) placing a normal tissue sample in an enzyme-free nuclear isolation medium comprising a cell nucleus-separating amount of a nonionic surfactant in a buffered aqueous solution that is substantially isotonic with the cell nuclei and has a pH (a) in the range of 5 to 8, and (b) is substantially the same as the physiological pH of the animal from which the tissue was taken, and separating a first group of single nuclei from the remaining normal tissue and cellular debris; (2) placing a suspected tissue sample in said nuclear isolation medium and separating a second group of single nuclei from the remaining suspected tissue and cellular debris; and (3) comparing the first group of single nuclei with the second group of nuclei.
Claim 24
Independent Claim
148 words
The method of comparing normal cell nuclei with suspected cell nuclei both said nuclei of the same histological type, said method comprising the steps of: (1) placing a normal tissue sample in an enzyme-free nuclear isolation medium comprising a cell nucleus-separating amount of a nonionic surfactant in a buffered aqueous solution that is substantially isotonic with the cell nuclei and has a pH (a) in the range of 5 to 8, and (b) is substantially the same as the physiological pH of the animal from which the tissue was taken, and separating a first group of single nuclei from the remaining normal tissue and cellular debris; (2) placing a suspected tissue sample in said nuclear isolation medium and separating a second group of single nuclei from the remaining suspected tissue and cellular debris; and (3) comparing the first group of single nuclei with the second group of nuclei.
Claim 25
Dependent Claim
33 words
The comparison method of claim 20 wherein the nuclear isolation medium contains a fluorescent, phosphorescence, or absorbance stain and the groups of cell nuclei are compared by analytical cytology analysis in step (3).
Claim 26
Dependent Claim
34 words
The comparison method of claim 20 wherein the nuclear isolation medium also contains serum albumin in an amount substantially the same as the physiological state of the animal from which the samples were taken.
Claim 27
Dependent Claim
38 words
The method of claim 22 wherein the nuclear isolation medium additionally contains magnesium and/or calcium ions in an amount substantially equal to the amounts of said ions in the fluid surrounding the nuclear membrane of the tissue sample.
Claim 28
Dependent Claim
27 words
The comparison method of claim 20 wherein the normal tissue sample is taken from one individual and the suspected tissue sample is taken from a second individual.
Claim 29
Dependent Claim
14 words
The method of claim 20 wherein said suspected cell nuclei are cancer cell nuclei.
Claim 30
Independent Claim
90 words
A method of dissociating cell nuclei while simultaneously staining for antigens, receptors or enzymes comprising the steps of (a) placing a tissue sample in a nuclear isolation medium containing an isotonic solution of a cell separating amount of a nonionic surfactant and at least one stain for antigens, receptors or enzymes; (b) physically dissociating the tissue sample while in contact with the nuclear isolation medium; and (c) allowing the dissociated tissue sample to remain in contact with the nuclear isolation medium until a quantity of single nuclei have been separated.
Claim 31
Independent Claim
90 words
A method of dissociating cell nuclei while simultaneously staining for antigens, receptors or enzymes comprising the steps of (a) placing a tissue sample in a nuclear isolation medium containing an isotonic solution of a cell separating amount of a nonionic surfactant and at least one stain for antigens, receptors or enzymes; (b) physically dissociating the tissue sample while in contact with the nuclear isolation medium; and (c) allowing the dissociated tissue sample to remain in contact with the nuclear isolation medium until a quantity of single nuclei have been separated.
Claim 32
Dependent Claim
14 words
A method as in claim 26 wherein the receptors are estrogen and progesterone receptors.
Claim 33
Dependent Claim
17 words
A method as in claim 26 wherein at least one of the stains is an antibody stain.
Claim 34
Independent Claim
102 words
A method of removing intact nuclei from cells containing same which are cultured on a tissue culture plate comprising contacting said cells with a nuclear isolation medium for dissociating a tissue or cell sample into discrete, non-agglomerated nuclei, the medium comprising a solution of a nonionic surfactant present in an amount sufficient to separate the cell nuclei while maintaining nuclear integrity dissolved in an aqueous solution that is substantially or completely isotonic with and maintains the normal electronic volume of the cell nuclei, under conditions such that said nuclei are released from said cells in a single step, and removing said cells.
Claim 35
Independent Claim
102 words
A method of removing intact nuclei from cells containing same which are cultured on a tissue culture plate comprising contacting said cells with a nuclear isolation medium for dissociating a tissue or cell sample into discrete, non-agglomerated nuclei, the medium comprising a solution of a nonionic surfactant present in an amount sufficient to separate the cell nuclei while maintaining nuclear integrity dissolved in an aqueous solution that is substantially or completely isotonic with and maintains the normal electronic volume of the cell nuclei, under conditions such that said nuclei are released from said cells in a single step, and removing said cells.
Claim 36
Dependent Claim
61 words
A nuclear isolation medium for dissociating a tissue or cell sample into discrete, non-agglomerated nuclei, the medium consisting of a solution of a nonionic surfactant present in an amount sufficient to separate the cell nuclei while maintaining nuclear integrity dissolved in an aqueous solution that is substantially or completely isotonic with and maintains the normal electronic volume of the cell nuclei.
Claim 37
Dependent Claim
61 words
A nuclear isolation medium for dissociating a tissue or cell sample into discrete, non-agglomerated nuclei, the medium consisting of a solution of a nonionic surfactant present in an amount sufficient to separate the cell nuclei while maintaining nuclear integrity dissolved in an aqueous solution that is substantially or completely isotonic with and maintains the normal electronic volume of the cell nuclei.
Claim 38
Dependent Claim
136 words
A nuclear isolation medium for cytometric analysis used to dissociate an animal tissue or cell sample into discrete, non-agglomerated cell nuclei, said medium consisting essentially of a nonionic surfactant present in an amount sufficient to separate the cell nuclei while maintaining nuclear membrane integrity; a microscopy stain for staining the DNA cell content; and divalent cations in an amount substantially equal to the amounts of said cations in the fluid surrounding the nuclear membrane of the tissue or cell sample, dissolved in an aqueous solution that is substantially or completely isotonic with and maintains the normal electronic volume of the cell nuclei, the medium having a pH that is in the range of pH 6.5 to 7.5 and is substantially identical to the physiological pH of the animal from which the tissue or cell sample originates.
Claim 39
Dependent Claim
136 words
A nuclear isolation medium for cytometric analysis used to dissociate an animal tissue or cell sample into discrete, non-agglomerated cell nuclei, said medium consisting essentially of a nonionic surfactant present in an amount sufficient to separate the cell nuclei while maintaining nuclear membrane integrity; a microscopy stain for staining the DNA cell content; and divalent cations in an amount substantially equal to the amounts of said cations in the fluid surrounding the nuclear membrane of the tissue or cell sample, dissolved in an aqueous solution that is substantially or completely isotonic with and maintains the normal electronic volume of the cell nuclei, the medium having a pH that is in the range of pH 6.5 to 7.5 and is substantially identical to the physiological pH of the animal from which the tissue or cell sample originates.
Claim 40
Dependent Claim
130 words
A nuclear isolation medium for DNA flow cytometric analysis used to dissociate an animal tissue or cell sample into discrete, non-agglomerated cell nuclei, said medium consisting essentially of a nonionic surfactant present in an amount sufficient to separate the cell nuclei while maintaining nuclear membrane integrity; a microscopy stain for staining the DNA cell content; and serum albumin in an amount substantially the same as the physiological state of the tissue or cell sample; dissolved in an aqueous solution that is substantially or completely isotonic with and maintains the normal electronic volume of the cell nuclei, the medium having a pH that is in the range of pH 6.5 to 7.5 and is substantially identical to the physiological pH of the animal from which the tissue or cell sample originates.
Claim 41
Dependent Claim
130 words
A nuclear isolation medium for DNA flow cytometric analysis used to dissociate an animal tissue or cell sample into discrete, non-agglomerated cell nuclei, said medium consisting essentially of a nonionic surfactant present in an amount sufficient to separate the cell nuclei while maintaining nuclear membrane integrity; a microscopy stain for staining the DNA cell content; and serum albumin in an amount substantially the same as the physiological state of the tissue or cell sample; dissolved in an aqueous solution that is substantially or completely isotonic with and maintains the normal electronic volume of the cell nuclei, the medium having a pH that is in the range of pH 6.5 to 7.5 and is substantially identical to the physiological pH of the animal from which the tissue or cell sample originates.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1993-03-05 | 1993-09-04 | 1994-03-06 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-03-05 | 1997-09-04 | 1998-03-06 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-03-05 | 2001-09-04 | 2002-03-06 | $7700.00 | Window Closed / Lapsed |