The TOP Flash and FOP Flash plasmids used in this study were purchased from Millipore (USA) and the pRLSV40 plasmid (Promega, USA) containing the Renilla reniformis luciferase gene (RLuc) under the control of the SV40 virus promoter (kindly provided by Professor Bu in the Peking University First Hospital Central Laboratory) was used as control. The expressing plasmids pcDNA 3.1(+)-H19 and pcDNA 3.1(+) empty vector were purchased from Genepharma (China). Cells were transfected with 500 ng H19 or empty vector, 500 ng TOP Flash or FOP Flash vector, and 12 ng RLuc using Lipofectamine 3000.12 h after transfection, cells were incubated with indicated doses of 1,25(OH)2D3 or equal quantities of ethanol for 48 and the analysis of Luc and RLuc activities was performed using the Dual Luciferase Reporter Assay System (Promega, USA) and the Luc and Rluc activities were measured with Synergy H2 microplate reader (Bio Tek Instruments, USA). The Luc activity was then normalized to RLuc activity.
Synergy h2 microplate reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy H2 microplate reader is a multipurpose instrument designed for absorbance, fluorescence, and luminescence detection. It features dual monochromators for flexible wavelength selection and offers a wide dynamic range to support a variety of microplate-based assays.
Automatically generated - may contain errors
Lab products found in correlation
El406 washer dispenser, by Agilent Technologies (2 mentions)
Fopflash, by Merck Group (1 mentions)
Pcdna3.1 empty vector, by GenePharma (1 mentions)
Prl sv40 plasmid, by Promega (1 mentions)
Pcdna3.1 h19, by GenePharma (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
10 protocols using synergy h2 microplate reader
1
Dual Luciferase Assay for H19 lncRNA
2
Paracellular Permeability Quantification
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Paracellular permeability was estimated via FD-4 flux. Briefly, Caco-2 cells were seeded in a 12-well Transwell system to reach monolayers. After treatment with AD and DSS, cells were incubated in the upper chamber with Hank’s balanced salt solution for 2 h, which contains 1 mg/mL FD-4 solution. FD-4 signal was determined via Synergy H2 microplate reader (Biotek Instruments, USA).
3
Intestinal Permeability Measurement in Mice
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Intestinal permeability of mice was measured as previously described with little modifications [29 (link)]. At 24 h postoperation, mice were administered intragastrically with FD-4 (150 mg/mL, Sigma, USA) for 4 μL/g body weight. 400 μL blood samples were collected at 4 h after the administered of FD-4. A Synergy H2 microplate reader (BioTek Instruments, USA) with 492 nm excitation and 520 nm emission filters was used to measure the serum fluorescence level. Concentration of FD-4 was measured using standard curve produced by continuous dilution of FD-4.
4
Multiplex Serum Biomarker Profiling
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Peripheral venous blood was drawn into pyrogen‐free tubes without any additives. After coagulation at room temperature, tubes were centrifuged at 1500 g for 10 min, and serum was stored at –80°C in multiple aliquots. Serum levels of CRP (Cat. no. DY1707), PTX3 (Cat. no. DY1826), sTNFR1 (Cat. no. DY225), OPG (Cat. no. DY805), DLL1 (Cat. no. DY1818), CXCL16 (Cat. no. DY1164), Axl (Cat. no. DY154), ePCR (Cat. no. DY2245), endostatin (Cat. no. DY1098), GDF‐15 (Cat. no. DY957), CatS (Cat. no. DY1183), CD147 (Cat. no. DY972), CCL18 (Cat. no. DY394), Gal3BP (Cat. no. DY2226), sCD163 (Cat. no. DY1607) and sCD166 (Cat. no. DY656) were measured in duplicate by enzyme‐linked immunosorbent assay with antibodies obtained from R&D Systems (Minneapolis, MN, USA) in a 384 format using a combination of a CyBi SELMA (CyBio, Jena, Germany), EL406 washer/dispenser (Biotek, Winooski, VT, USA) and Synergy H2 microplate reader (Biotek). vWF was measured by the same method with antibodies obtained from DakoCytomation (Glostrup, Denmark). The intra‐ and interindividual coefficients of variation were <10%.
5
Evaluating Bacterial Growth Regulation
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To determine the effect of toxin or TA loci expression on growth, cultures of E. coli strain BL21 (DE3) harboring pDEST527 plasmids were adjusted to 106 CFU/mL (OD600 = 0.001) in LB media with or without 1 mM IPTG. For expression from pBAD33 plasmids, cultures were adjusted to 106 CFU/mL (OD600 = 0.001) in LB media with or without 0.4% arabinose. Each culture was distributed into three wells of a 96-well plate. Cultures were grown in 200 μL volumes at 37 °C under continuous orbital shaking in a Synergy H2 microplate reader (Biotek, Winooski, VT, USA) and OD600 was measured every 20 min. For the analysis of E. amylovora CTBT3-1 harboring pEVS143 plasmids, cultures were adjusted to 106 CFU/mL (OD600 = 0.001) in LB media with or without 1 mM IPTG and OD600 was measured every 45 min at 28 °C. For all assays, at least three independent experiments were performed.
6
Intestinal Permeability Measurement in Mice
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Mice intestinal permeability was detected as mentioned previously [14] (link). On the 7th day of the animal experiment, FD-4 (150 g/L, Sigma, America) was administered to mice by gavage at a dose of 40 μL/10 g body weight. 4 h after FD-4 gavage, we collected the blood samples and detected the serum fluorescence level with Synergy H2 microplate reader (Biotek Instruments, America) with 492 nm excitation and 520 nm emission filter.
7
Melanin Quantification from Cell Lysates
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HEMs were lysed by sonication in radioimmunoprecipitation assay (RIPA) buffer (0.1 M Tris-HCl (pH 7.2) containing 1% Nonidet P-40, 0.01% sodium dodecyl sulfate, and a protease inhibitor cocktail; Roche Applied Science) and centrifuged at 15,000× g for 10 min. Protein concentrations in culture supernatants were quantified using a BCA Protein Assay Kit (Pierce; Thermo Fisher Scientific). Pellets containing melanin were dissolved in 1 N NaOH and incubated for 30 min at 60 °C. Protein concentrations and melanin contents were determined by measuring the absorbance at 562 nm and 450 nm, respectively, using a Synergy H2 microplate reader (BioTek). The melanin contents were normalized to the protein concentrations.
8
Quantification of LBP and CRP by ELISA
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Enzyme‐linked immunosorbent assay (ELISA) was used to determine the levels of LBP and C‐reactive protein (CRP) (cat# DY870 and DY1707, RnD systems, Minneapolis, MN) in a set‐up that combined a CiBi SELMA (CiBio, Jena, Germany), an EL406 washer/dispenser (Biotek, Winooski, VT), and a Synergy H2 microplate reader (Biotek). Optical density (OD) at 450 nm was used for quantification by comparison to a dilution series of a standard provided by the manufacturer. OD at 540 nm was used for wavelength correction. The intra‐ and interassay coefficients of variation were less than 10% for both assays.
9
FD-4 Paracellular Permeability Assay
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Paracellular permeability was measured using a previous method [19 (link), 20 (link)]. After the treatment described beforehand, monolayers were washed with phosphate-buffered saline (PBS) solution, and then 1 mg/mL FD-4 solution diluted by Hank's balanced salt solution was added to the apical compartments for 2 h. After taking 100 μL solution from the basolateral compartments, in 492 nm excitation and 520 nm emission filter, the fluorescence of FD-40 flux was assessed with Synergy H2 microplate reader (BioTek Instruments Inc., Winooski, VT, USA). Besides, calibration curves were drafted by serial dilution of FD-40 to determine FD-40 concentrations.
10
Measuring Intestinal Permeability with FD-4
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After intestinal epithelial barrier model was successfully constructed, LPS was added in the Apical side of the model and cultured with the supplementary of 1 mg/mL FD-4 solution for 2 h. The paracellular permeability was measured by FD -4 flux. A Synergy H2 microplate reader (Bio Tek) was used for the determination of FD-4 signaling.
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