Methods for staining cells for identification and storing

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 . A method for distinguishing sperm based on DNA content, comprising:
 staining sperm with a DNA-selective fluorescent dye by missing the sperm with the dye at a temperature substantially equal or less than 39° C.;   mixing the sperm with at least one nanoparticle;   electroporating the sperm, the dye and the at least one nanoparticle for a period of time to provide substantially uniform staining and concomitantly to substantially preserve sperm viability;   exposing the sperm to a light source to cause the stained DNA to fluoresce;   detecting a pre-determined fluorescence of the stained DNA, the pre-determined fluorescence corresponding to DNA content;   sorting the sperm based on the pre-determined fluorescence; and   collecting selected sperm from the sorted sperm.   
     
     
         2 . The method of  claim 1 , wherein the pre-determined fluorescence corresponds to a desired chromosome, chromosome fragment, an insertion or a deletion. 
     
     
         3 . The method of  claim 1 , wherein the dye comprises at least one selected from the group consisting of SYTOX blue, SYTOX green, SYTOX orange, a cyanine dimer, POPO-1, BOBO-1, YOYO-1, TOTO-1, JOJO-1, POPO-3, LOLO-1, BOBO-3, YOYO-3, TOTO-3, a cyanine monomer, PO-PRO-1, BO-PRO-1, YO-PRO-1, TO-PRO-1, JO-PRO-1, PO-PRO-3, LO-PRO-1, BO-PRO-3, YO-PRO-3, TO-PRO-3, TO-PRO-5, acridine homodimer, 7-amino actinomycine D, ethidium bromide, ethidium homodimer-1, ethidium homodimer-2, ethidium nonazide, nuclear yellow and propidium iodide. 
     
     
         4 . The method of  claim 1 , wherein the dye comprises at least one selected from the group consisting of SYTO 40 blue-fluorescent nucleic acid stain, SYTO 41 blue, SYTO 42 blue, SYTO 43 blue, SYTO 44 blue, SYTO 45 blue, a green-fluorescent SYTO dye, SYTO 9 green, SYTO 10 green, SYTO BC green, SYTO 13 green, SYTO 16 green, SYTO 24 green, SYTO 21 green, SYTO 27 green, SYTO 26 green, SYTO 23 green, SYTO 12 green, SYTO 11 green, SYTO 20 green, SYTO 22 green, SYTO 15 green, SYTO 14 green, SYTO 25 green, an orange-fluorescent SYTO dye, SYTO 83 orange, SYTO 84 orange, SYTO 85 orange, a red-fluorescent SYTO dye, SYTO 64 red, SYTO 61 red, SYTO 17 red, SYTO 59 red, SYTO 62 red, SYTO 60 red, SYTO 63 red, a Hoechst dye, Hoechst 33342, Hoechst 34580, Hoechst 33258, DAPI, LDS 751 and dihydroethidium. 
     
     
         5 . The method of  claim 2 , wherein the mixing is at a temperature between about −4° C. to about 30° C. 
     
     
         6 . The method of  claim 1 , wherein the mixing period of time is about 1 minute to about 15 minutes. 
     
     
         7 . The method of  claim 1 , wherein the mixing period of time is less than 1 minute. 
     
     
         8 . The method of  claim 1 , wherein the nanoparticle comprises a quantum dot or metallic nanoparticle. 
     
     
         9 . The method of  claim 1 , wherein the nanoparticle comprises a targeting molecule. 
     
     
         10 . The method of  claim 9 , wherein the targeting molecule binds DNA or a fluorescent dye. 
     
     
         11 . The method of  claim 1 , further comprising eliminating dead sperm before sorting the sperm. 
     
     
         12 . The method of  claim 1 , wherein the sperm are sorted by X- or Y-chromosome DNA content with at least 90% efficiency. 
     
     
         13 . The method of  claim 1 , wherein the viability of the sperm before sorting is at least 30%. 
     
     
         14 . An apparatus which distinguishes sperm based on DNA content, wherein the sperm are stained with DNA-selective fluorescent dye, comprising:
 means for suspending the sperm in a hypertonic condition to partially dehydrate the sperm, at a temperature substantially sufficient to maintain a comparatively high sperm viability rate, and for a time period less than 15 minutes;   means herein for transferring the sperm to a hypotonic condition at said same temperature;   wherein the dye is present in at least one of the hypertonic or hypotonic condition to expedite permeation of the dye into the sperm when osmotic equilibrium is reached, thereby staining DNA in the sperm;   a light source which exposes the sperm to light, which causes the stained DNA to fluoresce;   a detector which detects a per-determined fluorescence of the stained DNA, the predetermined fluorescence corresponding to DNA content;   means for killing the sperm based on the pre-determined fluorescence; and   means for collecting selected sperm.   
     
     
         15 . A method for breaching a cellular membrane of a cell to introduce a nanoparticle into the cell in an osmotic gradient process, comprising:
 incubating the live cell in a buffer-containing first solution in a hypertonic condition to partially dehydrate the cell, at a temperature between about 0° C. and about 12° C., which is substantially sufficient to maintain a comparatively high cell viability rate, and for a time period less than about 5 minutes;   transferring the solution containing the cell to a buffer-containing second solution in a hypotonic condition at said same temperature between about 0° C. and about 12° C.; and   wherein the nanoparticle is present in at least one of the hypertonic or hypotonic condition to expedite permeation of the nanoparticle into the cells when osmotic equilibrium is reached.   
     
     
         16 . The method of  claim 15 , wherein an incubation temperature of the cell is less than or equal to about 4° C. 
     
     
         17 . The method of  claim 15 , wherein the cell viability rate is greater than 70%. 
     
     
         18 . The method of  claim 15 , wherein the nanoparticle is present in the second solution in the hypotonic condition and the nanoparticle is drawn into the cell across the cellular membrane. 
     
     
         19 . The method of  claim 15 , further comprising:
 collecting the cell from a donor and subjecting the cell to extension prior to incubation; and   cooling the cell to 6° C.   
     
     
         20 . The method of  claim 19 , further comprising:
 cooling the collected cell after the collecting step, to about 4° C.;   subjecting the collected cell to a second extension; and   freezing the collected cell.