Nuclear isolation medium and procedure for separating cell nuclei
OwnerJerry T. Thornthwaiteinventor
Life of this patent
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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.
Claims32
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.
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.
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.
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.
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.
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.
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.
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.
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.
Dependencies are inferred from the claim text.