The antioxidant property of MBGCe_Gens was estimated as their ability to remove H2O2, one of the most significant ROS species. In correlation with the role of enzyme Catalase, the property is named CAT activity. The tests were performed using the Fluorimetric Hydrogen Peroxide Assay Kit from Sigma Aldrich with a TECAN GeniosPro microplate reader, as presented in a previous paper [16 (link)] Again, measurements were performed in SBF to simulate the physiological environment surrounding the implant. The presence of H2O2 in SBF is detected through its reaction with a molecular probe catalyzed by the peroxidase enzyme, which generates a red fluorescent product that can be analyzed fluorometrically. CAT activity is reported as the percentage of H2O2 decomposed at the end of the assay. We suspended 40 mg of MBGCe_Gen in 400 μL of 50 μM solution of H2O2 in SBF and measured the residual concentration of H2O2 after 120 min of soaking.
Genios pro microplate reader
Manufactured by Tecan
Sourced in Switzerland, Germany, Austria
The GENios Pro microplate reader is a versatile laboratory instrument designed for various absorbance-based applications. It is capable of performing multi-mode detection, including absorbance, fluorescence, and luminescence measurements, across a wide range of microplates.
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Lab products found in correlation
Axiovert 200m, by Zeiss (4 mentions)
Bradford assay, by Bio-Rad (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Cathepsin d activity assay kit, by Abcam (2 mentions)
Vivaspin 10 000 mwco, by Sartorius (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Fluorimetric hydrogen peroxide assay kit, by Merck Group (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Pmir report luciferase vector, by Thermo Fisher Scientific (1 mentions)
Dual light luciferase β galactosidase reporter gene assay system, by Thermo Fisher Scientific (1 mentions)
Quickchange multi site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Amplex red kit, by Thermo Fisher Scientific (1 mentions)
Synergy ht multi mode microplate reader, by Agilent Technologies (1 mentions)
Luciferase assay system, by Promega (1 mentions)
Prl cmv, by Promega (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Spark multimode, by Tecan (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
44 protocols using genios pro microplate reader
1
Antioxidant Efficacy of MBGCe_Gen
2
BODIPY-Cholesterol Efflux Assay
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BODIPY-cholesterol efflux assay was determined as previously described [27 (link)]. Briefly, a stock solution of BODIPY cholesterol was prepared at 5 mM in DMSO. HepG2 cells were loaded with 2.5 mM of BODIPY-cholesterol in culture medium for 2 h at 37 °C. Cells were rinsed twice with physiological buffer (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgSO4, 5 mM glucose, 20 mM HEPES, pH 7.4), and incubated with the same buffer for 1 h at 37 °C with shaking at 50 rpm. The cell supernatant was centrifuged for 5 min at 6800g, and the BODIPY fluorescence intensity in the supernatants was measured using a TECAN Genios Pro Microplate Reader (Tecan US, Inc., Morrisville, NC, USA) at excitation 490 ± 10 nm and emission 535 ± 20 nm.
3
Viability Assay for B. burgdorferi
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To estimate the viability of B. burgdorferi, the SYBR Green I/propidium iodide (PI) assay was performed as described by Feng et al. [35 (link)]. Briefly, SYBR Green I 5 µL (100 × stock, Invitrogen, Waltham, MA, USA) and 5 µL propidium iodide (0.5 mM, Sigma, St. Louis, MO, USA) were added to each well and mixed thoroughly. The plates were incubated in the dark for 15 min at room temperature, with λex at 450 nm and λem at 535 nm (green emission) and 635 nm (red emission) for each well of the screening plate using a TECAN Genios Pro microplate reader (Männedorf, Switzerland). Meanwhile, B. burgdorferi suspensions (live and 70% isopropyl alcohol killed) at five different proportions of live: dead cells (0:10, 2:8, 5:5, 8:2, 10:0) were mixed and added to the wells of the 96-well plate. Then, the SYBR Green I/PI reagent was added to each of the five samples, and the green/red fluorescence ratios for each proportion of live/dead B. burgdorferi were measured using the same parameters on the TECAN Genios Pro microplate reader as above. Using least-square fitting analysis, the regression equation and regression curve of the relationship between the percentage of live bacteria and the ratios of green/red fluorescence were obtained. The regression equation was used to calculate the percentage of live cells in each well of the screening plate.
4
Cytotoxicity Evaluation of Extracts
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All the chemicals used in this study were obtained from Sigma-Aldrich Chemicals Company (Munich, Germany). Acetone, dichloromethane, ethanol, ethyl acetate, and hexane were used as the extraction solvents. Dimethyl sulfoxide was used for dissolving extracts. Drabkin's reagent was used in the haemoglobin content determination assay. Daunorubicin was used as the standard cytotoxic drug, and MTT was used as an indicator of cell viability. Roswell Park Memorial Institute (RPMI) medium was used for growing the mammalian cells in vitro. A Celestron digital light microscope (Celestron Electronics, California, USA) and a haemocytometer were used in the determination of peritoneal cell count. All incubations were done in a Shel Lab incubator (Sheldon Manufacturing, Inc., Cornelius, USA), and for analysis of cell density, a GeniosPro microplate reader (Tecan Group Ltd, Mӓnnedorf, Switzerland) was used.
5
Antimicrobial Efficacy of Plant Extracts
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The antibacterial activity of the extracts was assessed using the broth microdilution method [17 ]. The root bark extracts were tested against Staphylococcus aureus NCTC 6571 (Culture Collections, Public Health England, Salisbury, UK). Stock solutions were prepared by dissolving the extracts in dimethyl sulfoxide (DMSO). Two‐fold dilutions of the extracts in nutrient broth were dispensed in a 96-well microplate. Overnight liquid cultures of S. aureus cells were adjusted to 0.5 McFarland's standard to make a final concentration of 2 × 106 CFU/mL. The inoculum was placed in each well and incubated at 37°C for 24 hours in an incubator (Lab DoctorTM, MIDSCI Co., Valley Park, USA). Ciprofloxacin was used as a positive control while a mixture of nutrient broth and DMSO was used as a negative control. The bacterial suspension without extracts was used as the growth control. Preincubation readings were recorded at 590 nm using a Genios Pro microplate reader (Tecan Group Ltd. Zurich, Switzerland). Postincubation readings were recorded after 24 h and the cell viability test was carried out by the MTT assay. The assay involved the addition of the MTT dye to each well, incubation for at least 2 h, and then observation of colour changes.
6
Luciferase reporter assay for miRNA target genes
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Wild-type (wt) and mutant (mut) seed regions of the target genes (PTEN, TXNIP, TSC1 and CAV1) were constructed and cloned into Luc 3′UTR between the HindIII and BcuI sites of pMIR-REPORT-Luciferase vector (Ambion by Thermo Fisher Scientific, Grand Island, NY, USA). The oligonucleotide sequences are listed in Supplementary Table S2 . Constructed vectors were verified by Sanger sequencing using Applied Biosystems® 3500 analyzer (Applied Biosystems, Foster City, CA, USA). AGS cells (100,000 cells/well) were co-transfected with 146 ng of constructs (wt or mut vector) and with 50 nM of either miRNA mimic or negative mimic control using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). After 48 h incubation luciferase activity was detected by Dual-Light™ Luciferase & β-Galactosidase Reporter Gene Assay System (Applied Biosystems, Foster City, CA, USA) following manufacturer’s protocol. Luminescent signal was quantified by GENios Pro microplate reader (Tecan Trading AG, Mannedorf, Switzerland). Reporter activity was normalized to β-Galactosidase activity.
7
SMAD4 Alternative Promoter Luciferase Assay
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Luciferase reporter constructs were cloned using the restriction map of the BAC729G3 bacterial artificial chromosome (BAC) clone from the RPCI-11 human BAC library (Invitrogen, Carlsbad, CA, USA), which covers the alternative promoter region of SMAD4 (Luc-371) [24 (link),36 (link)]. All SMAD4 constructs (Luc-371, Luc-216, and Luc-41) were confirmed using sequencing. Twenty-four hours after the transfection, cells were lysed with luciferase assay buffer. Luciferase activity was measured using the dual-luciferase reporter assay system following the manufacturer’s protocols (Promega, Madison, WI, USA), and luminescence was measured using a GENios Pro Microplate Reader (Tecan Trading AG, Mannedorf, Switzerland).
8
Validating HMGA2-miR-495 Interaction
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The interaction between HMGA2 3′UTR and miR-495 was predicted by TargetScanHuman 7.0 (www.targetscan.org ). To test the binding site of HMGA2 and miR-495, five bases in HMGA2 3′UTR were mutated using QuickChange Multi Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA), and dual-luciferase reporter assay was performed on the wildtype (wt) or mutant (mt) HMGA2 3′UTR in BGC-823 and HGC-27 cells using the kit Dual-Luciferase Reporter Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The intensity of fluorescence signals was detected by a GENios Pro microplate reader (Tecan, Männedorf, Switzerland).
9
Kinetics of P. aeruginosa Killing by Ethyl Acetate Extract
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The rate of kill of P. aeruginosa by the ethyl acetate extract was determined using a modified method from Sim et al. [28 (link)]. Extracts and bacteria used were prepared as described previously for determination of antibacterial activity [29 (link)]. Extract concentrations in the range of 12.5–100 µg/ml were separately added to the wells of a 96-well microplate with an equal volume of the standardised bacterial inoculum. Preincubation absorbance readings of the plate were measured at 590 nm using a microplate reader (Tecan GENios Pro microplate reader, Grödig, Austria). The cell density was then measured at 590 nm at 30-minute intervals for the first two hours, followed by two-hour intervals for 8 hours using a microplate reader (Tecan GENios Pro microplate reader, Grödig, Austria). The profiles of killing and regrowth of bacteria were assessed as a function of both time and extract concentrations.
10
GPR6-mediated β-Arrestin2 Recruitment Assay
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The GPR6 mediated β-arrestin2 recruitment was measured by using the PathHunter™ Chinese hamster ovary (CHO)-K1 β-arrestin2 human GPR6 eXpress kits. In this cell line, GPR6 receptors are fused with a β-galactosidase N-terminal fragment termed ProLink 1 (GPR6-PK1), and β-arrestin2 are fused to an N-terminal deleted version of β-galactosidase (EA-β-arrestin2). Activation of the receptor induces β-arrestin2 recruitment, causing complementation of the two β galactosidase enzyme fragments. Levels of the active enzyme are the direct result of β-arrestin2 recruitment caused by receptor activation and quantified using the PathHunter™ detection reagent containing β-galactosidase substrates. The assays were performed following manufacturer's instructions for the PathHunter eXpress kits. Briefly, cells were plated in 384-well plates in DiscoverX cell plating reagent1 for 48 hours in a humidified atmosphere at 37 °C and 5% CO2. Cannabinoids were serially diluted in cell plating reagent1. After incubation with ligand at 37 °C following manufacturer's instructions, the cells were incubated with detection reagent for 1 hour at room temperature in the dark. Subsequently, the chemiluminescence signal, measured as relative luminescence units, was detected using a TECAN GENios Pro microplate reader.
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