BACE1 enzymatic activity was determined in a homogeneous time-resolved fluorescence (HTRF) assay, and BACE2 enzymatic activity was determined in an enzyme-linked immunosorbent assay (ELISA)-based assay using an APP-derived peptide that contains the K670N + M671L (Swedish) double mutation of the APP β secretase cleavage site as a substrate. The recombinant human BACE1 (R&D Systems, MN, USA) was incubated (2 h at 25 °C) with the substrate and the inhibitor (9 or 10 concentrations at 1/5 steps from 10 μM). Fluorescence intensity was measured (excitation wavelength: 320 nm, measuring wavelength: 620 nm and 665 nm) using RUBYstar (BMG LABTECH, Ortenberg, Germany). The recombinant human BACE2 ectodomain was incubated (1 h at 37 °C) with the substrate and the inhibitor (10 concentrations at 1/4 steps from 25 μM). The count of chemi-luminescence in each well was measured using ARVOTM MX 1420 Multilabel Counter (PerkinElmer, Shelton, CT, USA). The inhibitory potential of the compounds on the enzymatic activity of BACE1 and BACE2 was determined by performing concentration–response curves.
Arvo mx 1420 multilabel counter
Manufactured by PerkinElmer
Sourced in United States
The ARVO MX 1420 multilabel counter is a versatile laboratory instrument designed for high-throughput detection and quantification of various types of samples, such as fluorescence, luminescence, and absorbance. The device is capable of performing a wide range of assays, including cell-based, biochemical, and molecular biology applications. The ARVO MX 1420 provides accurate and reliable results, making it a valuable tool for researchers and scientists in various fields of study.
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Lab products found in correlation
Recombinant human bace 1, by R&D Systems (1 mentions)
Rubystar, by BMG Labtech (1 mentions)
Zetasizer nano s, by Malvern Panalytical (1 mentions)
Ir affinity 1 spectrometer, by Shimadzu (1 mentions)
P 2100 polarimeter, by Jasco (1 mentions)
Varian vns500 spectrometer, by Agilent Technologies (1 mentions)
Jms t100lp accutof lc plus 4g spectrometer, by JEOL (1 mentions)
J 725 spectropolarimeter, by Jasco (1 mentions)
Quattro premier xe, by Waters Corporation (1 mentions)
Uv 1280 spectrophotometer, by Shimadzu (1 mentions)
Prism software, by GraphPad (1 mentions)
Imark microplate absorbance reader, by Bio-Rad (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Cck 8, by PerkinElmer (1 mentions)
Pll coated 96 well plates, by Greiner (1 mentions)
Cell counting kit 8 solution, by Dojindo Laboratories (1 mentions)
Tracp alp assay kit, by Takara Bio (1 mentions)
Costar 3915, by Corning (1 mentions)
14 protocols using arvo mx 1420 multilabel counter
1
BACE1 and BACE2 Enzymatic Inhibition Assays
2
Quantifying Cell Invasion using FluoroBlok Assay
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The cell invasion was measured using a Corning® BioCoatTM FluoroBlokTM24-Multiwell Insert System (Corning, Corning, NY, USA). GFP-SAS cells were treated with siRNAs for 24 h, after which they were trypsinized, counted, and normalized for cell number between treatments. Cells (1 × 105) in serum-free DMEM were placed in an insert made of polycarbonate membrane with 8-μm pores and precoated with basement membrane matrix. The outer chamber was filled with 0.5 ml of DMEM containing 5% FBS. The plate was incubated at 37°C for 24 hours. The fluorescence of the invaded cells was read at 485/535 nm with ARVOTM MX 1420 Multilabel Counter (PerkinElmer, Waltham, MA, USA).
3
Evaluating Virus Susceptibility in Transgenic Plants
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Evaluation of the virus infection in transgenic and non-transgenic plants was carried out by the direct double antibody sandwich form of ELISA (DAS-ELISA) procedure [73 (link)]. Equal amounts of fresh leaf tissue were sampled from the same areas and on the same day. Each treatment was tested separately 3 weeks after viral inoculation for virus susceptibility using anti-CP polyclonal antibody (Japan Plant Protection Association) to detect PVY and rabbit polyclonal antibody (https://orders.agdia.com/agdia-set-pva-alkphos-sra-60000 ) to detect PVA. Each well’s optical density was read at 405 nm with a plate reader (ARVO MX 1420 Multilabel Counter, Perkin-Elmer). The absorbance values were corrected by subtracting the average triplicate absorbance readings for each sample and the average buffer blanks triplicate readings. Virus-infected samples were those with the mean absorbance values higher than R (the mean ± standard deviation for triplicate independent of the negative control samples) [74 (link)]. Blank, mock-inoculated (WT/Mock and StAPI5-OE/Mock), and virus-resistant potato cv. Degima inoculated with PVY and PVA separately (Resistant/PVY and Resistant/PVA) plants were grown as negative controls while the virus-inoculated potato wild-type (WT/PVY and WT/PVA) as positive controls.
4
Characterization of Silica Nanoparticles
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Rhodamine-labeled silica NPs, 25 and 115 nm in diameter, with hydroxyl groups on the surface (neutral surface charge; N) or functionalized with amino modified (positive surface charge; +q) or carboxyl modified (negative surface charge; –q) were purchased from HiQ-Nano, Arnesano, Italy. All NPs were suspended in water at 25 mg/L and then dispersed with 0.3× Danieau’s solution (17.4 mM NaCl, 0.21 mM KCl, 0.18 mM Ca(NO3)2, 0.12 mM MgSO4, and 1.5 mM HEPES buffer, pH 7.6). The particle size distribution was determined with a nano-zetasizer (Zetasizer Nano S; Malvern Instruments, Worcestershire, UK), which uses DLS technique [39 (link)]. Hydrodynamic sizes of all NPs dispersed in the embryo medium at the highest concentration used for the treatment were analyzed from the time of exposure up to 24 h in order to check the stability of the NP solutions during the contact with embryos. The fluorescence intensities of three types of silica NPs were measured at different concentrations using an ARVO™MX 1420 Multilabel Counter (Perkin Elmer, Waltham, MA, USA). The successive dilutions of the medium at particle concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 mg/L were completed immediately prior to exposure.
5
Spectroscopic Characterization of Chemical Compounds
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Optical rotation was recorded on a JASCO P-2100 polarimeter (Jasco Co., Tokyo, Japan). UV spectrum was recorded on a Shimadzu UV-1280 spectrophotometer (Shimadzu Co., Kyoto, Japan). ECD spectrum was recorded on a JASCO J-725 spectropolarimeter. IR spectrum was recorded on a Shimadzu IR affinity-1 spectrometer. NMR spectra were recorded on an Agilent Varian VNS500 spectrometer (Agilent Technologies, Santa Clara, CA, USA). Chemical shifts (ppm) were referenced to the residual solvent peaks (δH 3.31 and δC 49.0 for CD3OD). Negative-mode ESITOFMS was obtained on a JEOL JMS-T100LP AccuTOF LC-plus 4 G spectrometer using a sample dissolved in MeOH (Jeol Ltd., Tokyo, Japan). For LC-MS/MS analysis, Quattro Premier XE (Waters Co., Milford, MA, USA) system was used. Optical densities were measured using a microplate reader (ARVO MX 1420 multilabel counter, Perkin Elmer).
6
Enzymatic Cleavage Kinetics of Fluorogenic Substrates
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Each fluorogenic substrate was dissolved in dimethylformamide at a concentration of 10 mM. Since our previous studies demonstrated that ASP shows sufficient proteolytic action in phosphate buffer solution (pH ranging from 7.2 to 7.5) [17 (link)–19 (link)], in the present study we mixed 2 μl of the substrate solution (10 mM) with 148 μl of ASP or ASP[R566A] (1 nM) in 20 mM sodium phosphate buffer (pH 7.4) in order to examine the cleavage of each substrate by ASP or ASP[R566A]. The mixture was incubated at 37°C for 30 min. After incubation, the reaction was stopped by adding 150 μl of acetic acid.
As the substrate releases 7-amino-4-methyl-coumarin by receiving the cleavage at the P1 site (the bond following Lys-Lys), we determined the proteolytic activity of ASP or ASP[R566A] by measuring the fluorescence generated. That is, the fluorescence of the solution was measured with at λex = 340 nm and λem = 440 nm. A spectrofluorometer (ARVO MX 1420 Multilabel Counter, Perkin Elmer, San Jose, CA, USA) was used. Estimations of maximum velocity (Vmax) and the Michaelis-Menten constant (Km) were performed by using nonlinear regression analysis within GraphPad Prism software (GraphPad Software Inc., La Jolla, CA).
As the substrate releases 7-amino-4-methyl-coumarin by receiving the cleavage at the P1 site (the bond following Lys-Lys), we determined the proteolytic activity of ASP or ASP[R566A] by measuring the fluorescence generated. That is, the fluorescence of the solution was measured with at λex = 340 nm and λem = 440 nm. A spectrofluorometer (ARVO MX 1420 Multilabel Counter, Perkin Elmer, San Jose, CA, USA) was used. Estimations of maximum velocity (Vmax) and the Michaelis-Menten constant (Km) were performed by using nonlinear regression analysis within GraphPad Prism software (GraphPad Software Inc., La Jolla, CA).
7
Evaluating Cell Growth and Viability
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To evaluate cell growth and viability, dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium (MTS) assays were performed using CellTiter 96 Aqueous One-Solution cell proliferation assay (Promega, Madison, WI, USA). Huh7 cells were incubated with 1 mL of fresh DMEM supplemented with 10% FBS containing 0%, 0.1%, 0.5%, 1%, 5% and 10% miso extracts. PXB cells were incubated with 1 mL of fresh dHCGM containing 0%, 0.1%, 0.5%, 1%, 5% and 10% miso extracts. After 24 hours of treatment with or without miso extracts, absorbance at 490 nm of each well was measured with an iMark Microplate Absorbance Reader (Bio-Rad) or an ARVO MX 1420 multilabel counter (PerkinElmer, Boston, MA, USA).
8
FGF18 Impacts on Cell Proliferation
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M0-Mø or RAW264.7 cells (5 × 103 cells/well) were seeded on the 96-well plate and incubated with complete DMEM medium overnight. The complete DMEM medium supplemented with FGF18 at 1 or 10 ng/ml was added to each well at 0 h. At 24, 48, 72 or 96 h of culture, cell counting kit-8 (CCK-8, Dojindo, Japan) solution was added and incubated for 2 h at 37°C following manufacturer’s instruction. Then, incubated CCK-8 solution was collected, and the absorbance was measured at 450 nm using an ARVO™ MX 1420 multilabel counter (PerkinElmer, USA). This experiment was performed in triplicate and repeated at least three times (n = 3–4).
9
Quantification of Cellular NAD+/NADH Levels
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Fluorescent NAD/NADH detection kit (Cell Technology, USA) was used for quantification of intracellular NADH and NAD+ concentration following the manufacturer’s protocol. For each extraction, 1 mL of culture broth (3 days after IPTG induction) was collected in a 1.5 mL microfuge tube and subsequently centrifuged at 16,000×g for 1 min at 4 °C. After removal of the supernatant, the resulting cell pellet was immediately immersed in liquid nitrogen. Next, the pellet was resuspended in 100 μL of the NAD+ or NAD(H) extraction buffer and 100 μL of the NAD/NADH lysis buffer. After the samples were heated at 60 °C for 20 min, 100 μL of the reaction buffer and 200 μL of the opposite (NAD(H) or NAD+) extraction buffer were added into the tube. The lysate was mixed and centrifuged at 8,000 g for 5 min at 4 °C. The supernatant was then retrieved for NAD+ and NADH analysis. The supernatant was added with the enzyme mix and fluorescent NADH reaction reagent according to the manufacturer’s instructions. The mixture was incubated at room temperature under a dark condition for 90 min. Readings were taken using an ARVO MX 1420 multilabel counter (PerkinElmer) with excitation at 544 nm and emission at 590 nm.
10
Mexiletine-Induced Ion Channel Assay
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Cells were seeded onto poly-l-lysine (PLL)-coated 96-well plates (Greiner Bio-One, Kremsmünste, Austria) at 8000-10,000 cells/well with Micro Shot 706 (MSTechnos, Tokyo, Japan). They were cultured in DMEM supplemented with 10% FBS and 300-600 µM mexiletine for 48-72 h. The quantity of the culture medium was unified with 100 μL. A calculated volume of baculoviral vector solution was directly added to cells in the wells approximately 24 h before the assay. On the assay, the cells were washed with the bathing solution using Multi Works 508 (MSTechnos) in the following manner. Two hundred microliters of the bathing solution was slowly poured into the wells, and then 200 μL of the solution was removed. This procedure was repeated six times for washout of mexiletine. After the washout, 100 μL of bathing solution that contains test compounds and BaCl 2 was added to each well to bring the final concentration of Ba 2+ to 200 μM. Cells in 96 wells were incubated for 12-16 h at 37 °C, and then proceeded to MTT assay. Plates were analyzed for absorption on ARVO MX (1420 Multilabel Counter; PerkinElmer, Waltham, MA).
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