Spectramax gemini em microplate reader

ATP hydrolysis capacity of CtUba4 WT and mutants in the presence of CtUrm1 protein was measured using the fluorometric pyrophosphate assay kit (Abcam). The reactions were performed in a buffer composed of 100 mM MES pH 6.0; 100 mM NaCl; 0.5 mM MgCl₂; and 20 μM ATP at 37°C for 30 min. Final CtUba4 and CtUrm1 concentrations were 5 and 7.5 μM, respectively. Reaction mixtures were centrifuged in 30K Amicon Ultra‐0.5 centrifugal filter devices at 4°C for 30 min at 19,000 g in order to separate the pyrophosphate molecules from the protein components. Flow‐through samples were used to measure pyrophosphate concentrations, according to manufacturer's guidelines. The fluorescence was measured using a SpectraMax^® Gemini EM Microplate Reader (Molecular Devices). Three to five independent experiments were performed, each with two technical replicates.

Cell migration assays were performed using FluoroBlok™ 96-Multiwell Insert Plates with 3.0 µm High Density PET Membrane (#351161, Thermo Fisher Scientific, Waltham, MA, USA) as described before (10 (link)). The CD11b⁺Gr1⁺ MDSCs were isolated using the EasySep™ Mouse Myeloid-Derived Suppressor Cell Isolation kit (#19867, Stemcell Technologies, Cambridge, MA, USA) according to the manufacturer’s instructions. The isolated cells were prelabeled with 5 μM of calcein AM (#564061, BD Biosciences, San Jose, CA, USA) and then were seeded in the upper chamber at the density of 1 × 10⁵/50 µl/insert. Normal MLFs were cultured with or without (control) TGFβ treatment (10 ng/ml) for 24 h. The media containing residual TGFβ were removed, and after washing, fresh media were added. Conditioned media (CMs) were collected after an additional 48 h. The control or TGFβ-treated MLF CMs were incubated with B7H3 blocking antibody (15 µg/ml) or isotype IgG and then added to the lower chamber of the plates. The plates were incubated for 18 h followed by fluorescence measurement using SpectraMax Gemini EM Microplate Reader (Molecular Devices, San Jose, CA, USA).

Cell viability was analyzed using the alamar blue assay. The assay indicates the cellular metabolic activity, which depends on the cell viability and on the number of cells (proliferation) in the culture. A549 cells were exposed to particle suspensions (in DMEM⁺), corresponding to total Ni concentrations of 0.1, 1, 5, 10, 20 and 40 μg cm^-2, for 24 and 48 h in transparent 48 well plates. These concentrations equal to 0.1, 1, 5, 10, 20 and 40 μg mL^-1. After exposure, the suspensions were removed and the cells were incubated in 200 μL of 10% alamarBlue^® (Invitrogen, Life Technologies) for 3 h. Fluorescence was measured using 560 nm excitation and 590 nm emission wavelengths (Molecular Devices SpectraMax^® Gemini EM Microplate Reader). Each experiment was performed three times in duplicate wells. Possible interferences between the particles and alamar blue were examined with a similar assay at cell-free conditions. Cell viability studies were also performed with the released fraction of Ni (S1 File, S2 Fig). In addition, cell viability was analyzed in terms of the cell membrane integrity with the trypan blue exclusion assay, as described by Midander and co-workers [17 (link)]. For this assay, the cells were exposed for 4 h to each particle type at a total Ni concentration of 20 μg cm^-2.

Both assays were performed from a single sample of 200 mg of PM muscle that was crushed in phosphate buffer 50 mM (KH₂PO₄ 50 mM, K₂HPO₄ 50 mM, pH 7.0) with an Ultra-Turrax (IKA, Staufen, Germany) and centrifuged 30 min at 10°C to collect supernatant that was further used for the two assays. For cytochrome oxidase activity (COX), enzymatic reaction was done from 100 μg of proteins in phosphate buffer (KH₂PO₄ 50 mM and K₂HPO₄ 50 mM, pH 7.0) in the presence of cytochrome 0.1 mM, n-dodecylmaltoside 0.15%, and DTT 5 mM (Sigma, Saint-Quentin-Fallavier, France). The reduction of cytochrome c was measured at 550 nm using a Tecan M200 microplate reader (Tecan, Lyon, France). For reactive oxygen species (ROS) assay, 100 μg of proteins was diluted in a reaction buffer (250 mM sucrose, 50 mM KCl, 25 mM Tris-base, 10 mM K₂HPO₄, pH 7.4). The oxidation of the 2,7-dichlorodihydrofluorescein diacetate 5 mM by oxygen reactive species was measured at 528 nm using a Spectramax Gemini EM microplate reader (Molecular Devices, Sunnyvale, CA, United States).

A PicoProbe acetyl-CoA fluorometric assay kit (BioVision) and a SpectraMax Gemini EM Microplate Reader (Molecular Devices) were used to measure acetyl-CoA amount.