Methods for staining cells for identification and sorting

Abstract

The present invention provides novel methods of cell staining, such as bovine sperm, using electroporation or osmolality treatments at viability-enhancing temperatures. Furthermore, methods of highly efficient cell sorting that are especially suitable in sorting bovine sperm using novel cell staining procedures are also provided.

1 - 20 . (canceled) 
     
     
         21 . A method for labeling DNA in a plurality of cells using a tagged oligonucleotide, comprising:
 (a) incubating the plurality of cells in a first solution, at a temperature from about −4° C. to about 39° C., wherein the first solution is a hypertonic solution having an osmolality of at least about 250 mOsm;   (b) forming a mixture comprising the plurality of cells, the tagged oligonucleotide, and a second solution, wherein the second solution is a hypotonic solution having an osmolality of less than about 250 mOsm, and wherein at least the second solution comprises a buffer compatible with electroporation;   (c) incubating the mixture, at a temperature from about −4° C. to about 39° C.; and   (d) electroporating the mixture to provide substantially uniform labeling of DNA.   
     
     
         22 . The method of  claim 21 , wherein the tagged oligonucleotide specifically hybridizes to the DNA at a DNA sequence complementary to the tagged oligonucleotide, wherein the DNA sequence complementary to the tagged oligonucleotide is a selected chromosomal region, and hybridization of the tagged oligonucleotide and the DNA sequence complementary to the tagged oligonucleotide allows subsequent detection of the selected chromosomal region. 
     
     
         23 . The method of  claim 22 , wherein the plurality of cells are mammalian. 
     
     
         24 . The method of  claim 23 , wherein the plurality of cells are bovine sperm. 
     
     
         25 . The method of  claim 24 , wherein duration of incubation in the first solution is about less than 15 minutes. 
     
     
         26 . The method of  claim 24 , wherein the osmolality of the first solution is between about 500 mOsm and about 730 mOsm, and the osmolality of the second solution is between about 150 mOsm and 250 mOsm. 
     
     
         27 . The method of  claim 24 , wherein the tagged oligonucleotide comprises a fluorescent label. 
     
     
         28 . The method of  claim 27 , wherein the tagged oligonucleotide further comprises a quantum dot or a nanoparticle. 
     
     
         29 . The method of  claim 27 , wherein the fluorescent label is selected from the group consisting of fluorescein isothiocyanate, fluorescein dichlorotriazine, fluorinated analogs of fluorescein, naphthofluorescein carboxylic acid, naphthofluorescein carboxylic acid succinimidyl ester, carboxy rhodamine 6G, pyridyloxazole derivative Cy2, pyridyloxazole derivative 3, pyridyloxazole derivative C5, phycoerythrin, fluorescent species of Succinimidyl esters, carboxylic acids, isothiocyanates, sulfonyl chlorides, or dansyl chlorides, propionic acid Succinimidyl esters, pentanoic acid Succinimidyl esters, Succinimidyl esters of carboxytetramethylihodamine, rhodamine Red-X succinimidyl ester, Texas Red sulfonyl chloride, Texas Red-X succinimidyl ester, Texas Red-X sodium tetrafluorophenol ester, Red-X: Texas Red dyes, tetramethylrhod amine, lissarnine rhodamine B, tetramethylrhodamine, tetramethylrhodamine isothiocyanate, naphthofluorescein, coumarin derivatives, pyreries, pyridyloxazole derivatives, dapoxyl dyes, Cascade Blue dye, Cascade Yellow dye, benzofuran isothiocyanates, sodium tetrafluorophenols, and 4.4-difluoro-4-bora-3a,4a-dia-Za-S-indacene. 
     
     
         30 . The method of  claim 24 , wherein incubating the mixture prior to electroporation is at a first metabolically-suspending temperature for a duration of about less than 15 minutes. 
     
     
         31 . The method of  claim 30 , wherein the first metabolically-suspending temperature is about 0° C. to about 4° C. 
     
     
         32 . The method of  claim 24 , further comprising:
 (e) incubating the mixture after electroporation at a second metabolically-suspending temperature for a duration of about less than 15 minutes.   
     
     
         33 . The method of  claim 32 , wherein the second metabolically-suspending temperature is about 0° C. to about 4° C. 
     
     
         34 . The method of  claim 24 , wherein electroporation is a pulse of about 0.25 seconds. 
     
     
         35 . The method of  claim 24 , wherein the first solution comprises a buffer compatible with electroporation. 
     
     
         36 . The method of  claim 24 , wherein the tagged oligonucleotide is present in the first solution. 
     
     
         37 . The method of  claim 21 , wherein the tagged oligonucleotide is present in at least the second solution to permeate the membrane of each of the plurality of cells when osmotic equilibrium with the second solution is reached.