| Solution Information | help | |
| Enzyme: | G-protein coupled receptor 55 | |
| inhibitor: | BDBM76084 | |
| 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 GPR55 receptor 3) Culture Media: MEM with L-glutamine, Pen-strep, 10% Fetal Bovine Serum and selection antibiotics - 100ug/ml G418 and 50ug/ml Zeocin 4) Agonist Working Solution: Lysophosphatidylinositol (Avanti Polar #850090P, 5mM stock in DMSO) diluted to 50uM in water. 5) Control Working Solution: 100% DMSO (for positive control wells: no agonist will be added to mimic 100% inhibition of agonist-mediated response; for negative control wells: LPI agonist will be added to mimic 0% inhibition of agonist-mediated response). 6) Test Compounds 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) 45 ul of cell suspen | |
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