| Assay Method Information | |
| | In Vitro Activity Assay |
| Description: | 1. Experimental Steps1) Solution Preparation10% BSA (Bovine Serum Albumin)10 g of BSA was dissolved in 100 mL of double-distilled water (ddH2O) to obtain 10% BSA.10 mM ODQ Stock Solution1 mg of ODQ powder was weighed and dissolved in 534 μL of DMSO to prepare 10 mM ODQ solution. The solution was aliquoted and stored in a −20° C. refrigerator. Washing Buffer (50 mL) Volume Final concentration49 mL of Earls balanced 1xsalt solution (EBSS)500 μL of 1M hydroxyethyl piperazine 10 mMethanesulfonic acid (HEPES)250 μL of 10% BSA stabilizer 0.05%250 μL of 1M MgCl2 5 mMAssay Buffer (50 mL)Volume. Final concentration 48.95 mL of Earls balanced 1xsalt solution (EBSS)500 μL of 1M hydroxyethyl piperazine 10 mMethanesulfonic acid (HEPES)250 μL 10% BSA 0.05%50 μL of 500 mmol/L 0.5 mM isobutylmethylxanthine (IBMX)250 μL of 1M MgCl2 5 mM Detection Buffer) 50 μL of cGMP-D2 (D2-labeled cyclic guanosine monophosphate) was added to 1 mL of lysis buffer and mixed thoroughly.b) 50 μL of anti-cGMP cryptate (Eu3+ cryptate-labeled anti-cyclic guanosine monophosphate antibody) was added to 1 mL of lysis buffer and mixed thoroughly.2) Compound Dilution(1) The compound was diluted to a concentration of 10 mM using DMSO.(2) A serial dilution of the compound was performed, resulting in 10 different concentration levels of each compound. 100 nL of each diluted compound was added to a 96-well plate.3) Preparation of LNCap Cells(1) LNCap culture medium: RPMI1640 supplemented with 10% fetal bovine serum (FBS) and 1% double antibody(2) The phosphate-buffered saline (PBS), trypsin, and culture medium for use in cell passage were preheated in a 37° C. water bath.(3) Cells were removed from a 37° C., 5% Co2 incubator, and the old culture medium was removed from the culture flask using a pipette.(4) 5 mL of PBS was pipetted into the flask for rinsing the cells, and then the liquid was discarded.(5) 3 mL of trypsin was pipetted into the flask. After shaking, the liquid was discarded, and the flask was returned to the incubator.(6) Approximately 2 minutes later, the flask was retrieved, and cell detachment was observed. 9 mL of culture medium was pipetted into the culture flask and mixed by repetitive pipetting. The cell suspension was then transferred to a 50 mL centrifuge tube.(7) 0.7 mL of cell suspension was pipetted into a counting chamber, and cell counting was performed using the ViCell XR. The remaining cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded.(8) 10 mL of washing buffer was added to wash the cells, followed by another centrifugation at 1000 rpm for 5 minutes, after which the supernatant was discarded.(9) Assay buffer was added, and the cell concentration was adjusted to 3×106/mL. |
| Affinity data for this assay | |
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