Method for using division arrested cells in screening assays
View Patent ↗Division arrested cells are used in screening assays to determine the effect of a substance of interest on the cells. The division arrested cells can be used in drug screening assays, signal transduction assays, and are especially useful in large scale, high throughput assays.
1. A method for determining if a substance of interest has an effect on a G-protein coupled receptor (GPCR) signal in a division arrested mammalian cell, the method comprising:
(a) measuring the signal in the division arrested mammalian cell after the cell is contacted with the substance of interest and comparing the signal with a signal measured when the cell has not been contacted with the substance of interest, and
(b) determining whether the substance of interest has an effect on the GPCR signal in the division arrested mammalian cell by identifying a comparative difference in the signal when the division arrested mammalian cell has and has not been contacted with the substance of interest,
wherein the division arrested mammalian cell is a mammalian cell in which the meiotic or mitotic cycle has been stopped and in which cellular division can no longer take place.
2. The method of claim 1 , wherein activation of the G-protein coupled receptor is measured.
3. The method of claim 1 , wherein the G-protein coupled receptor is overexpressed by the division arrested mammalian cell.
4. The method of claim 1 , wherein the G-protein coupled receptor is selected from the group consisting of:
(a) a serotonin receptor;
(b) a muscaranic acetylcholine receptor; and
(c) a β2 adrenergic receptor.
5. The method of claim 1 , wherein the GPCR signal measured in the division arrested mammalian cell results from Ca 2+ mobilization.
6. The method of claim 1 , wherein the GPCR signal measured in the division arrested mammalian cell is determined by measuring the activity of a reporter.
7. The method of claim 6 , wherein the reporter is selected from the group consisting of secreted alkaline phosphatase, luciferase, and green fluorescent protein.
8. The method of claim 1 , wherein the division arrested mammalian cell is a cell selected from the group consisting of:
(a) a Chinese hamster ovary cell;
(b) a NIH3T3 cell;
(c) a HEK293 cell; and
(d) a M1WT3 cell.
9. The method of claim 8 , wherein the cell is a Chinese hamster ovary cell.
10. The method of claim 1 , wherein the meiotic or mitotic cycle of the mammalian cell has been stopped by irradiation of the mammalian cell or exposure of the mammalian cell to mitomycin C.
11. The method of claim 10 , wherein the irradiation of the mammalian cell is gamma irradiation.
12. The method of claim 1 , wherein the substance of interest has efficacy as a GPCR agonist.
13. The method of claim 1 , wherein the substance of interest has efficacy as a GPCR antagonist.
14. The method of claim 1 , wherein the division arrested mammalian cell has been frozen and thawed before contact with the substance of interest.
15. The method of claim 14 , wherein the cell is a Chinese hamster ovary cell.
16. The method of claim 1 , wherein the division arrested mammalian cell has previously been gamma irradiated.