Assay-ready frozen cell and method for minimizing variability in the performance thereof
View Patent ↗The invention provides an assay-ready frozen eukaryotic cell (stored frozen) in a composition that includes a cryopreservative and an inhibitor of xanthine oxidase, a kit containing a plurality of the assay-ready frozen eukaryotic cell, and a method of preparation thereof to minimize the variability in the performance of the cell, particularly in terms of undesirable intra- and inter-assay variability.
1. An assay-ready eukaryotic cell stored frozen in a composition comprising a cryopreservative and allopurinol at a concentration in the composition in a range of about 50 to 200 μM.
2. The assay-ready frozen eukaryotic cell of claim 1 , wherein the concentration of allopurinol in the composition is in a range of about 75 to 150 μM.
3. The assay-ready frozen eukaryotic cell of claim 1 , wherein the concentration of allopurinol in the composition is about 100 μM.
4. The assay-ready frozen eukaryotic cell of claim 1 , wherein said cryopreservative is dimethylsulfoxide (DMSO).
5. The assay-ready frozen eukaryotic cell of claim 4 , wherein the amount of DMSO in the composition is about 10%.
6. The assay-ready frozen eukaryotic cell of claim 1 , wherein said cryopreservative is glycerol.
7. The assay-ready frozen eukaryotic cell of claim 1 , wherein said cryopreservative is a combination of about 2.5% dimethylsulfoxide (DMSO) and about 10% glycerol.
8. The assay-ready frozen eukaryotic cell of claim 1 , wherein the composition further comprises fetal bovine serum (FBS).
9. The assay-ready frozen eukaryotic cell of claim 8 , wherein the amount of FBS in the composition is about 40%.
10. The assay-ready frozen eukaryotic cell of claim 1 , wherein the composition further comprises a scavenger of reactive oxygen species.
11. The assay-ready frozen eukaryotic cell of claim 10 , wherein said scavenger of reactive oxygen species is N-acetylcysteine (NAC) or glutathione SH (GSH).
12. The assay-ready frozen eukaryotic cell of claim 11 , wherein the concentration of NAC is in a range of about 1 mM to 10 mM.
13. The assay-ready frozen eukaryotic cell of claim 11 , wherein the concentration of NAC is about 5 mM.
14. The assay-ready frozen eukaryotic cell of claim 11 , wherein the concentration of GSH is about 1 mM.
15. The assay-ready frozen eukaryotic cell of claim 1 , wherein allopurinol is at a concentration of about 100 μM and said composition further comprises about 40% FBS and a concentration of NAC in a range of about 2.5 mM to 5 mM.
16. The assay-ready frozen eukaryotic cell of claim 1 , containing a reporter gene construct comprising a nucleotide sequence encoding a reporter gene product operatively linked to one or more transcriptional control elements that is regulated by the signal transduction activity of a cell surface protein in response to an extracellular signal.
17. The assay-ready frozen eukaryotic cell of claim 1 , in which the cells were treated with an anti-mitotic or pro-apoptotic agent prior to being frozen for storage.
18. The assay-ready frozen eukaryotic cell of claim 1 , wherein the eukaryotic cell is selected from the group consisting of a mammalian, avian, fish, insect, and yeast cell.
19. The assay-ready frozen eukaryotic cell of claim 1 , wherein the eukaryotic cell is a human cell.
20. The assay-ready frozen eukaryotic cell of claim 18 , wherein the eukaryotic cell is a chicken cell.
21. The assay-ready frozen eukaryotic cell of claim 18 , wherein the eukaryotic cell is a zebrafish cell.
22. The assay-ready frozen eukaryotic cell of claim 19 , wherein the human cell is a promonocytic cell.
23. A kit for conducting an assay, comprising:
a testing device having a plurality of wells; and
a reagent comprising a plurality of the assay-ready frozen cell of claim 1 .
24. The kit of claim 23 , wherein said testing device is a microtiter plate.
25. The kit of claim 23 , wherein said reagent is disposed in the wells of said testing device.
26. A method for preparing the assay-ready frozen eukaryotic cell of claim 1 , comprising:
adding a composition comprising a cryopreservative and an inhibitor of xanthine oxidase to a eukaryotic cell; and
cooling the cell at a rate of about 1° C. per minute until about −80° C. for frozen storage of the assay-ready eukaryotic cell, thereby improving the performance, upon thawing, of the assay-ready cell in an assay.
27. The method of claim 26 , wherein, prior to adding the composition comprising a cryopreservative and an inhibitor of xanthine oxidase to the eukaryotic cell, the eukaryotic cell is treated with an anti-mitotic or pro-apoptotic agent and then the eukaryotic cell is removed from contact with the anti-mitotic or pro-apoptotic agent.
28. The method of claim 27 , wherein the anti-mitotic or pro-apoptotic agent is vinblastin.
29. The method of claim 26 , wherein a plurality of assay-ready frozen eukaryotic cells are prepared and stored frozen in a plurality of separate individual vials or containers and the method minimizes the variability between individual vials or containers in the performance of the assay-ready frozen eukaryotic cells, upon thawing, in an assay.