Surefire lysis buffer

AlphaScreen assays to measure phosphorylated ERK 1/2 and cAMP were performed in 48-well plates using cells transiently transfected with 100ng of the wild-type or mutant CaSR pEGFP-N1 constructs 48-hours prior to performance of assays. For phosphorylated ERK 1/2 studies, cells were incubated in serum-free media for 12 hours prior to 5-minute treatment with 0.1-10mM CaCl₂. Cells were then lysed in Surefire lysis buffer (Perkin Elmer), and phosphorylated ERK 1/2 and total ERK1/2 assays were performed as previously described (21 (link)). For the cAMP assays, cells were treated with 10μM forskolin for 30 minutes prior to CaCl₂ treatment in stimulation buffer [1x Hanks Buffered Saline Solution, 0.1% BSA, 0.1% 3-isobutyl-1-methylxanthine (IBMX), 0.5mM HEPES] plus 0.1-10mM CaCl₂. Cells were incubated for 15 minutes, then lysed in a HEPES-based solution (0.1% BSA, 0.3% Tween-20, 5mM HEPES, pH7.4) and incubated for 4 hours. The fluorescence signal in AlphaScreen assays was measured using the PHERAstar FS microplate reader (BMG Labtech) (48 (link)). Nonlinear regression of concentration-response curves was performed with GraphPad Prism for the determination of IC₅₀ (i.e., [Ca²⁺]_e required for 50% inhibition of the maximal response).

hDOPr FlpIn CHO cells (CHO-hDOPr) were seeded into 96-well plates at a density of 50 000 cells/well. After 5–7 h, cells were washed with phosphate buffered saline (PBS) and incubated overnight in serum-free DMEM. Initially, time-course experiments were conducted at least twice for each ligand to determine the time required to maximally promote ERK1/2 phosphorylation via the δ-receptor. Concentration–response experiments were performed for the orthosteric ligands in the absence or presence of increasing concentrations of the allosteric modulator at 37 °C. Stimulation of the cells was terminated by removal of the media and the addition of 100 μL of SureFire lysis buffer (PerkinElmer) to each well. The plate was shaken for 5 min at room temperature before transferring 5 μL of the lysates to a white 384-well Proxiplate (PerkinElmer). Then 8 μL of a 240:1440:7:7 mixture of Surefire activation buffer/Surefire reaction buffer/Alphascreen acceptor beads/Alphascreen donor beads was added to the samples and incubated in the dark at 37 °C for 1.5 h. Plates were read using a Fusion plate reader (PerkinElmer).