Tristar lb 941 plate reader

BRET was performed as described previously (17 (link), 25 (link), 88 (link)). Briefly, HEK293 cells were maintained in 1x Dulbecco’s Modified Eagle Medium (Mediatech, Inc) without phenol red and supplemented with 10% fetal bovine serum (FBS), 2 mM L-Glutamine, and 1% penicillin/streptomycin (VWR, Calbiochem, and Invitrogen, respectively). Cells were transiently transfected with polyethyleneimine and RGS14-Luciferase(Luc) plus Gαi1-YFP (89 (link)) for 40 h. Cell were resuspended in Tyrode’s Solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 0.37 mM NaH₂PO₄, 24 mM NaHCO₃, 10 mM HEPES, and 0.1% glucose, pH 7.4) and plated at 10⁵ cells per well in a 96-well Optiplate (PerkinElmer Life Sciences). YFP expression was quantified using a TriStar LB 941 plate reader (Berthold Technologies) at 485 nm excitation and 530 nm emission. Next, 5 μM coelenterazine H (Nanolight Technologies) was incubated for 2 min, and BRET measurements were taken at 485 nm (Luc emission) and 530 nm (YFP emission). The Net BRET ratio was quantified as follows: (530 nm signal/485 nm signal) – (485 nm signal from Luc alone). Acceptor/donor ratios were calculated as follows: (530 nm signal from YFP measurement)/(485 nm signal from BRET measurement). Each experiment was repeated three times.

We also used the YFP-Epac-RLuc plasmid to measure intracellular cAMP levels (Ji et al., 2017 (link)). YFP-Epac-RLuc was transfected into HEK293-GHSR1a, HEK293-OX1R and HEK293-GHSR1a/ OX1R cells. The cells were collected and distributed in a 96-well white microplate after 24 h and cultured in HEPES-buffered phenol red-free medium for another 24 h. Cells were washed with PBS and resuspended with Dulbecco’s phosphate buffered saline (D-PBS). BRET was measured at room temperature. Cells were stimulated with agonists (ghrelin 100 nM and/or orexin-A, 100nM) for 5 min. BRET readings were collected by Tristar LB941 plate reader (Berthold technologies GmbH & Co., Germany).

DLBCL cells were incubated on a 96-well plate with either full medium or medium with indicated inhibitors used at concentrations specified in figures and figure legends. After incubation, cell viability was assessed with the 3-(4, 5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega). IC₅₀ values were calculated using GraphPad Prism v6.0 software. Detection of apoptosis was performed with Annexin V-FITC Apoptosis Detection Kit BD Biosciences and analyzed using FACS Canto flow cytometer (BD Biosciences). Caspase activity was measured using Caspase 3/7 Glo assay (Promega). Briefly, 0.15 ×10⁶/mL cells were incubated overnight with either AD-O51.4 or TRAIL (0.1 nM). Thereafter, Caspase Glo reagent was automatically injected to wells and the luminescence was measured using TriStar LB 941 plate reader (Berthold Technologies).

Cytotoxicity of oprozomib and bortezomib was assayed by using the 2.5-diphenyltetrazolium bromide (MTT) assay. Briefly, 5 × 10⁴ cells (A549, pmLF) per well were seeded in 24-well plates or 2.5 × 10⁴ cells (mATII cells) were seeded in 48 well plates. The next day, cells were incubated with different concentrations of oprozomib or bortezomib for 72 hours (A549, pmLF) or 52 hours (mATII cells). Then, a solution of 5 mg thiazolyl blue tetrazolium bromide (Sigma) per milliliter PBS was added to each well and incubated at 37°C for 1 hour to allow reduction of the tetrazolium dye to its insoluble formazan within the cell. After aspiration of the supernatant, the blue formazan crystals were dissolved in isopropanol + 0.1% Triton X-100 (Life Technologies). Absorbance was measured at 570 nm in a Tristar LB 941 plate reader (Berthold Technologies).

Constitutive interactions between GPR103-OX1R and GPR103-OX2R, BRET measurements were performed as previously described22 (link). Briefly, HEK293 cells were transiently transfected with the following combinations: pRluc-OX1R and/or pEGFP-GPR103, pRluc-GPR103 and/or pEGFP-OX1R, pRluc-OX2R and/or pEGFP-GPR103, and pRluc-GPR103 and/or pEGFP-OX2R. Cells were stimulated with OR-a, OR-B or QRFP (Phoenix Pharmaceuticals, USA) for 15 min. Following washes, cells were re-suspended in PBS followed by the addition of 5 μM Coelenterazine h for BRET measurements using the Tristar LB941 plate reader (Berthold, Germany) with two filter settings (Rluc filter, 400 ~ 475 nm; and EGFP filter, 500 ~ 550 nm).

A solution of DiI-labelled VLPs at 2×10¹² VLPs ml⁻¹ was mixed at a 7 : 1 (v/v) ratio with each liposome suspension at 2.5 g lipids l⁻¹. Fluorescence was monitored for 200 s to ensure baseline stabilization prior to the addition of the fusion-inducing buffer [0.1 M sodium acetate (pH 5.2), or 0.1 M NaH₂/Na₂HPO₄ (pH 8.5) in the negative control]. Hemifusion was monitored for an additional 800 s before addition of a detergent (decyldimethylamine-N-oxide; Anatrace) to solubilize the viral membrane completely, as a measure of 100 % fusion. Fluorescence was monitored with a TriStar LB941 plate reader (Berthold Technologies). For pH threshold determination, SPG buffers (succinic acid, sodium dihydrogen phosphate and glycine mixed at a 2 : 7 : 7 molar ratio), titrated to various pH values between 4 and 9, were used to induce fusion. The reported pH values were determined by measuring the pH of the VLP and liposome suspension after detergent solubilization.