Solution Information | help | |
Enzyme: | G-protein coupled receptor 35 | |
inhibitor: | BDBM61650 | |
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 GPR35 receptor 3) Culture Media: MEM with L-glutamine, Pen-Strep, 10% Fetal Bovine Serum, 10mM Hepes, and selection antibiotics - 200ug/ml G418 and 50ug/ml Zeocin 4) Positive Control Working Solution: 2.0% DMSO diluted in PBS 5) Agonist Working Solution: Zaprinast (Alexis # ALX-430-020-M010 - 10mg, 5mM stock in DMSO) diluted in PBS to 100uM 6) Fixative Working Solution: 6% Paraformaldehyde (PFA) diluted in PBS. 7) Nuclear Stain Working Solution: DAPI (Invitrogen, D1306) diluted to 150ng/ml in DAPI buffer (10mM TRIS, 10mM EDTA, 100mM NaCl, pH 7.4). Primary Screen Procedure: 1) 45ul of cell suspension (133,000 cells/ml in culture medium) was dispensed in each well of the assay plates using a Wellmate bulk dispenser. 2) Plates were incubated overnight or approximately 20 hours at 37 degree C and 5% CO2. 3) Serum was removed | |
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