Spark multimode reader

Enzyme-linked Immunosorbent Assays (ELISAs) were performed with plasma samples using commercially available ELISA kits for Ghrelin (ThermoFisher, # BMS2192) and GIP (Merck, EZHGIP-54K) according to the manufacturer’s specifications. Absorption was determined at 450 nm in Tecan Spark^® Multimode Reader.

Reaction mixture (10 µL) containing 50 ng of mRNA, Ribolock RNase inhibitor (40 U), complete amino acids mixture (50 µM) and nuclease-treated rabbit reticulocyte lysate was incubated at 30 °C for 1.5 h in a thermal cycler with heated lid (65 °C). Translation reactions were stopped by addition of cycloheximide solution (100 µM, in DMSO). 1 µL aliquot of the quenched reaction mixture was combined with 20 µL of reconstituted Nano-Glo luciferase assay reagent prepared by combining Nano-Glo luciferase assay substrate and Nano-Glo luciferase assay buffer (1:50) according to manufacturer´s protocol. The mixture was incubated at room temperature for 3 min and 20 µL aliquots were transferred to white opaque 384-well microplate prior to luminescence measurements with 1000 ms integration time on Tecan Spark Multimode Reader. The data are reported as counts/sec. For negative control, mRNA was omitted.
Details for other methods are included in Supplementary Information file.

Luciferase activity was measured using the dual-luciferase reporter assay system (Promega). Plasmid pRL-TK (Promega) which expressed Renilla luciferase was utilized as an internal control to correct the differences in transfection. For transfection, 10 ng pRL-TK and 50 ng pGL3.0-basic/p-(−1.6k/+0.2k)/p-∆NF-κB were co-transfected into MLE-12 cells in a 48-well plate using Lipofectamine 3000 (Thermo). After 24 h, cells were treated with or without IL-1β (10 ng/ml). Cells were harvested 24 h later and luciferase activity was measured using Tecan Spark multimode reader (Switzerland).

MMP was determined using the cationic fluorescent redistribution dye tetramethylrhodamine, methyl ester (TMRM) (Thermo Fisher Scientific, Waltham, MA, United States) as previously described (Sun et al., 2016 (link)). Briefly, cells were incubated with a final concentration of 30 nM TMRM at 37°C for 20 min. Cells were then washed with PBS and the fluorescence was recorded using a Spark Multimode Reader (Tecan, Männedorf, Switzerland). MMP fluorescence was normalized to the protein concentration.

Free intracellular zinc levels were measured by using the cell permeable and zinc selective probe FluoZin-3 AM (Invitrogen, Darmstadt, Germany), as previously described [48 (link)]. The zinc-dependent fluorescence changes were recorded either in a kinetic measurement, starting immediately after adding the stimulants, by using a Spark multimode reader (temperature: 37 °C, excitation: 485 nm, emission: 535 nm) (Tecan, Crailsheim, Germany) or measured as end point determinations by using flow cytometry (FACSCalibur, BD Bioscience, Heidelberg, Germany). The intracellular zinc concentration was determined by the following formula: $\left[Z{n}^{2+}\right]={\mathrm{K}}_{\mathrm{D}}\times \frac{\mathrm{F}-{\mathrm{F}}_{\min }}{{\mathrm{F}}_{\max }-F}$ , using a dissociation constant (K_D) of 8.9 nM for the Zn/FluoZin-3 AM complex. F_max and F_min values were reached by incubating cells with 100 µM zinc sulfate in combination with 5 µM of its ionophore sodium pyrithione (F_max) or with 100 µM of its chelator N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) (Sigma-Aldrich) (F_min), respectively.

Cell culture plates were coated with 0.5 mg/ml rat tail tendon collagen type Ⅰ (Solarbio, C8062, Beijing, China) overnight at 4°C and washed with PBS for three times before cell seeding. Murine hepatocytes were isolated from C57BL/6 N mice by a retrograde perfusion of liver with 30–40 ml HBSS (Gibco, 14175–095) containing 0.5 mM EGTA (Sigma, 03777), followed with about 40 ml low glucose DMEM containing 100U/mL collagenase type IV (Gibco, 17104–019). Hepatocytes were cultured in DMEM with 10% fetal bovine serum for 4 h for attachment. Then the medium was replaced with hepatocyte culture medium (LONZA, CC-3199 and CC-4182). The hepatocytes were treated with APAP (5 and 10 mM) in the absence or presence of CSA (50 μM) or chloroquine (20 μM) for 24 h. Cells were double stained with calcein and propidium iodide (Beyotime Biotech, C2015). After incubation at 37°C for 30 min, the fluorescence intensity was detected by Tecan’s Spark multimode reader to assess the percentage of cell death. For representative images, hepatocytes were stained with propidium iodide and Hoechst 33258 (10 μg/ml) for 30 min followed by fluorescence microscopy.