Use of a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly in a staining or sorting process

• US 20050282245A1
• Filed: 03/29/2005
• Published: 12/22/2005
• Est. Priority Date: 03/29/2004
• Status: Active Grant

• Current Assignee: Inguran LLC
• Original Assignee: Monsanto Technology LLC (Bayer AG)
• Inventors: Crowley, Kathleen S., Graham, Jeffrey A., Ludwig, Cindy L., Anzar, Muhammad
• Granted Patent: US 7,998,700 B2
• Time in Patent Office: Days
• US Class Current: 435/40.5
• CPC Class Codes: C12N 5/0612 sorting of gametes, e.g. according to sex or motility
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Abstract

Staining mixtures comprising viable spermatozoa, a composition which regulates oxidation/reduction reactions intracellularly or extracellularly, and a DNA selective dye are disclosed. The cells contained in such suspensions tend to have a greater capacity for enduring the various process steps typically associated with the sorting of sperm cells into gender enriched populations, thereby resulting in post-sort compositions with an increased number of viable or motile sperm. Processes for staining sperm cells comprising the formation of a staining mixture are also disclosed.

Claims

1. A staining mixture comprising viable spermatozoa, a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly, and a DNA selective dye, the concentration of the composition in the staining mixture being greater than 50 μ

M when the composition is pyruvate.

2. The staining mixture of claim 1, wherein the composition is selected from the group consisting of pyruvate, vitamin K, lipoic acid, glutathione, flavins, quinones, superoxide dismutase, superoxide dismutase mimics, and any combinations thereof.

3. The staining mixture of claim 2, wherein the composition is selected from the group consisting of pyruvate, vitamin K, lipoic acid, and combinations thereof.

4. The staining mixture of claim 1, wherein the composition comprises pyruvate at a concentration selected from the group consisting of about 2.5 mM, about 10 mM, about 15 mM, about 25 mM,and about 50 mM.

5. The staining mixture of claim 1, wherein the composition comprises vitamin K at a concentration selected from the group consisting of about 10 μ

M, about 50 μ

M, about 75 μ

M, and about 100 μ

M.

6. The staining mixture of claim 1, wherein the composition comprises lipoic acid at a concentration selected from the group consisting of about 0.1 mM, about 0.5 mM, about 0.75 mM, about 1.0 mM,and about 1.5 mM.

7. The staining mixture of claim 1, wherein the DNA selective dye is a DNA selective fluorescent dye.

8. The staining mixture of claim 2, wherein the DNA selective dye is a DNA selective fluorescent dye.

9. The staining mixture of claim 1, wherein the dye is a UV excitable or a visible light excitable dye.

10. The staining mixture of claim 2, wherein the dye is a UV excitable or a visible light excitable dye.

11. The staining mixture claim 1, wherein the dye is selected from the group consisting of Hoechst 33342, Hoechst 33258, SYBR-14, and bisbenzimide-BODIPY®

conjugate 6-{[3-((2Z) -2-{[1-(difluoroboryl)-3,5-dimethyl-1H-pyrrol-2-yl]methylene}-2H-pyrrol-5-yl)propanoyl]amino}-N-[3-(methyl{3-[({4-[6-(4-methylpiperazin-1-yl)-1H,3′

H-2,5′

-bibenzimidazol-2′

-yl]phenoxy)acetyl)amino]propyl}amino)propyl]hexanamide.

12. The staining mixture claim 2, wherein the dye is selected from the group consisting of Hoechst 33342, Hoechst 33258, SYBR-14, and bisbenzimide-BODIPY®

conjugate 6-{[3-((2Z)-2-{[1-(difluoroboryl)-3,5-dimethyl-1H-pyrrol-2-yl]methylene}-2H-pyrrol-5-yl)propanoyl]amino}-N-[3-(methyl{3-[({4-[6-(4-methylpiperazin-1-yl)-1H,3′

H-2,5′

-bibenzimidazol-2′

-yl]phenoxy}acetyl)amino]propyl}amino)propyl]hexanamide.

13. A process for staining sperm cells, the process comprising forming a staining mixture containing intact viable sperm cells, a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly, and a DNA selective dye, the concentration of the composition in the staining mixture being greater than 50 μ

M when the composition is pyruvate.

14. The process of claim 13, wherein the DNA-selective dye is selected from the group consisting of Hoechst 33342, Hoechst 33258, SYBR-14, and bisbenzimide-BODIPY®

conjugate 6-{[3-((2Z)-2-{[1-(difluoroboryl)-3,5-dimethyl-1H-pyrrol-2-yl]methylene}-2H-pyrrol-5-yl)propanoyl]amino}-N-[3-(methyl{3-[({4-[6-(4-methylpiperazin-1-yl)-1H,3′

H-2,5′

-bibenzimidazol-2′

-yl]phenoxy}acetyl)amino)propyl}amino)propyl]hexanamide.

15. The process of claim 13, wherein the staining mixture is subjected to a temperature of about 4°

C. to about 30°

C.

16. The process of claim 14, wherein the staining mixture is subjected to a temperature of about 30°

C. to about 39°

C.

17. The process of claim 14, wherein the staining mixture is subjected to a temperature of about 40°

C. to about 50°

C.

18. The process of claim 13, wherein the composition comprises pyruvate at a concentration selected from the group consisting of about 2.5 mM, about 10 mM, about 15 mM, about 25 mM,and about 50 mM.

19. The process of claim 13, wherein the composition comprises vitamin K at a concentration selected from the group consisting of about 10 μ

M, about 50 μ

M, about 75 μ

M, and about 100 μ

M.

20. The process of claim 13, wherein the composition comprises lipoic acid at a concentration selected from the group consisting of about 0.1 mM, about 0.5 mM, about 0.75 mM, about 1.0 mM, and about 1.5 mM.