Optiplate

Amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) was performed using 384-well microtiter plates (white opaque OptiPlate^TM, Perkin Elmer, Waltham, MA) containing 2.5 μl of 1:100 diluted sera and 2.5 μl of GST or GST-fusion proteins (10 μg/ml) in AlphaLISA buffer (25 mM HEPES (pH 7.4), 0.1% casein, 0.5% Triton X-100, 1 mg/ml dextran-500, and 0.05% Proclin-300). The reaction mixture was incubated at room temperature for 6-8 h. Next, anti-human IgG-conjugated acceptor beads (2.5 μl of 40 μg/ml) and glutathione-conjugated donor beads (2.5 μl of 40 μg/ml) were added and incubated further for 7 days at room temperature in the dark. The chemical emission was examind using EnSpire Alpha microplate reader (Perkin Elmer). Specific reactions were calculated by subtracting Alpha values of GST control from the values of GST-fusion proteins.

G protein activation was assayed by BRET as previously described (24 (link)). Briefly, plasmids encoding G protein biosensors and receptors of interest were cotransfected into HEK293T cells. Forty-eight hours after transfection, cells were washed twice with PBS, detached and resuspended in PBS containing 0.1% (w/v) glucose at room temperature. Cells were then distributed (80 μg of proteins per well) in a 96-well microplate (Optiplate, PerkinElmer). BRET² between RLuc8 and GFP10 was measured 1 min after addition of 5 μM coelenterazine 400a/Deep blue C (Gentaur). BRET readings were collected using an Infinite F200 reader (Tecan). The BRET signal was calculated as the ratio of emission of GFP10 (510–540 nm) to RLuc8 (370–450 nm).

HEK-CYGYEL cells were washed and resuspended in DPBS, and then centrifuged in order to pellet the cells. DPBS was aspirated and cells were resuspended in buffer containing 5 mM Tris and 2 mM EDTA (pH 7.3), then sonicated on low power for three 10 s cycles at 4 °C. Lysates were then spun down at 21,000× g for 20 min at 4 °C, after which the supernatant was obtained and stored at −80 °C or used for BRET assays. During the assay, lysates were mixed with coelenterazine h (5 µM), distributed into a white, 384-well OptiPlate (PerkinElmer) with lysates corresponding to the equivalent of approximately 5000 cells per well, and then assayed as above using a PHERAstar FSX plate reader. Cell lysates were also used to generate spectral scans, using a CLARIOstar plate reader (BMG Labtech).

Cells were treated according to the outlined protocol. Whole-cell lysates were prepared as described and incubated with Caspase 3/7 substrate (BD Pharmingen) for 60 min at 37 °C in a 96-well plate (OptiPlate, Perkin Elmer) and protein concentration was determined by a Bradford Assay (Roth). Substrate cleavage was measured for 50 cycles with 10 s delay (excitation at 380 nm, emission at 430–460 nm, Victor X4, Perkin Elmer). Kinetics were measured and calculated to the amount of protein per sample. Cells were treated according to the outlined protocol. For PARP cleavage analysis the samples were precipitated by acetone, boiled in 3× SDS-sample buffer for 10 min at 95 °C, and subjected to SDS-PAGE and Western blot analysis, using anti-Actin C4 (Sigma) and PARP polyclonal (Cell Signaling).

Liposomes (4 mg) were made using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) 5:4:1 (w/w/w). The lipids were combined in a glass tube and blown down to dryness under N2. ANTS (12.5 mM) and DPX (45 mM) were dissolved in 10 mM Tris pH 7.4, 100 mM KCl solution. ANTS (500 μl) and DPX (500 μl) were added to the dry lipids, the mixture vortexed and rehydrated at 37 °C for 1 h. After 5 min in a sonication bath, the liposomes were passed 25 times through a mini-extruder containing a 400-nm polycarbonate filter. Unincorporated ANTS/DPX was removed using a Zeba Spin Desalting column (7 K Molecular weight cut-off, 5 ml; Thermo Scientific). Bile salts stocks and dilutions were made with 10 mM Tris pH 7.4, 100 mM KCl. Assays were performed in a 96-well white OptiPlate (PerkinElmer) and contained 5 μl of the ANTS/PDX liposomes mixed with 200 μl of bile salt of different concentrations. Reactions were incubated at room temperature for 1 h in the dark. The plate was read on Fusion reader (PerkinElmer) using the following settings: emission filter, 355 nm; excitation filter, 530 nm; 5 s read per well; and PMT voltage set to high.