| Solution Information | help | |
| Enzyme: | Kappa-type opioid receptor | |
| inhibitor: | BDBM75580 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay Materials: 1) 384-well plates, black with clear bottom (Greiner# 781091) 2) U2OS (Human Osteosarcoma) cell line stably expressing the Beta-arrestin GFP and the KOR1 receptor 3) Culture Media: MEM with L-glutamine, Pen-strep, 10% Fetal Bovine Serum and selection antibiotics - 200ug/ml G418 and 100ug/ml Zeocin 4) Positive Control Working Solution: (Dynorphin-A, American Peptide #24450 - 5mM stock in DMSO) diluted in water to 10uM. 5) Negative Control Working Solution: Naloxone hydrochloride dehydrate (Sigma N7758)- 5mM stock diluted to 5uM in water. 6) Test compounds from dry powder working solution: 10mM in 100% DMSO 7) Fixative Working Solution: 6% Paraformaldehyde (PFA) diluted in PBS. 8) Nuclear Stain Working Solution: DAPI (Invitrogen, D1306) diluted to 150ng/ml in DAPI buffer (10mM TRIS, 10mM EDTA, 100mM NaCl, pH 7.4). Assay Procedure: 1) 45ul of cell suspension (200,000 cells/ml in culture medium) was dispensed in each well of the assay plates using a Wellmate bulk disp | |
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