The cells were treated with different concentration of curcumol (0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125, 0.0015625, 0.00078125 mg/mL) and ribavirin (4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 mg/mL) in the 96-well plates (0.1 mL/well). At the same time, untreated group (no drug) was applied and were incubated at 37 °C and 5% CO2 for 72 h. The morphology of cells in the culture was observed every day and photographed. After 72 h of incubation, the supernatant was discarded, 0.02 mL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H- tetrazolium bromide (MTT, Solarbio, China) was added to each well and incubated for 4 h. Subsequently, the supernatant was discarded and 0.15 mL of DMSO was added to each well, and incubated for 30 min. The optical density (OD value) was measured at 490 nm using the plate type multi-function analyzer (Tristar 2 SLB 942, Berthold Technologies, Berthold Technologies). The cytotoxic ratio (CR) was expressed as CR = (OD control—OD test)/OD control. CC50 was calculated by nonlinear regression analysis using GraphPad Prism™ software 5.0 (GraphPad Software, Inc. California, USA) [37 (link)]. CC50 is the constituent concentration at which 50% of cells have developed lesions while MNTC is a maximal concentration of a constituent that enables at least 80% of cells to survive [38 (link)].
Tristar2 s lb 942
Manufactured by Berthold Technologies
Sourced in Germany
The TriStar2 S LB 942 is a high-precision automated gas sorption analyzer designed for the characterization of porous materials. It is capable of performing surface area and pore size analysis using various gas adsorption techniques.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Prism software 5, by GraphPad (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Solarbio (1 mentions)
Sigmaplot 13, by Grafiti LLC (1 mentions)
Fugene hd, by Promega (1 mentions)
Luciferin substrate, by Promega (1 mentions)
Luminol l 012, by Fujifilm (1 mentions)
Horseradish peroxidase, by AppliChem (1 mentions)
E2129, by Merck Group (1 mentions)
Quantikine hs elisa kit, by R&D Systems (1 mentions)
Prism 10, by GraphPad (1 mentions)
Disruptor genie, by Scientific Industries (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Butyrylcholinesterase assay kit, by Nanjing Jiancheng (1 mentions)
22 protocols using tristar2 s lb 942
1
Cytotoxic Effects of Curcumol and Ribavirin
2
Kinetic Analysis of Marine Luciferases
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The kinetic parameters of the BL according to the coelenterazine (CTZ) analogues were determined with Lineweaver– Burk equation (Table 1 ).
A series of reaction solutions were prepared beforehand. Firstly, the CTZ variants were dissolved in HEPES buffer (50 mM, pH 7.2) to be 0.02–100 µM, and deployed in a 96-well black-frame optical-bottom microplate. Secondly, the recombinant marine luciferases (RLuc8.6-535 and ALuc16) were also dissolved in the same HEPES buffer to be 0.2 µM, and primed in the automatic injectors of a microplate reader (TriStar2 S LB942, Berthold), respectively. The substrates were placed in the microplate (final concentrations: 0.01–50 µM) beforehand and the microplate was then set on the sample stage of the microplate reader. The marine luciferase (final concentration: 0.1 µM) primed in the reader injector were injected into each well of the microplate and the corresponding BL intensities were recorded every 0.1 s during the initial five seconds. The final concentrations of the substrates were at 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, and 100 µM. The measurements were quadruplicated for the following statistical analyses (n = 4). The Km and Vmax values were calculated from Lineweaver–Burk plots using the Enzyme Kinetics Wizard in the commercially available SigmaPlot 13.0 software package (Systat Software Inc., San Jose, CA).
A series of reaction solutions were prepared beforehand. Firstly, the CTZ variants were dissolved in HEPES buffer (50 mM, pH 7.2) to be 0.02–100 µM, and deployed in a 96-well black-frame optical-bottom microplate. Secondly, the recombinant marine luciferases (RLuc8.6-535 and ALuc16) were also dissolved in the same HEPES buffer to be 0.2 µM, and primed in the automatic injectors of a microplate reader (TriStar2 S LB942, Berthold), respectively. The substrates were placed in the microplate (final concentrations: 0.01–50 µM) beforehand and the microplate was then set on the sample stage of the microplate reader. The marine luciferase (final concentration: 0.1 µM) primed in the reader injector were injected into each well of the microplate and the corresponding BL intensities were recorded every 0.1 s during the initial five seconds. The final concentrations of the substrates were at 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, and 100 µM. The measurements were quadruplicated for the following statistical analyses (n = 4). The Km and Vmax values were calculated from Lineweaver–Burk plots using the Enzyme Kinetics Wizard in the commercially available SigmaPlot 13.0 software package (Systat Software Inc., San Jose, CA).
3
Luciferase Assay for Cell Lines
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C33A cells (7 × 104) or primary mouse keratinocytes were transfected using Fugene HD (Promega) and DNA amounts indicated in the figure legends. Firefly and renilla luciferase activities were determined 48 h post transfection using a TriStar2 S LB 942 multimode plate reader (Berthold Technologies).
4
Serum BChE and Amyloid-Beta Assays
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Serum BChE activity was detected using a BChE assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Levels of Aβ1-42 and Aβ1-40 were examined with ELISA kits (Meimian, Yancheng, China). A multifunctional microplate reader (TriStar2S LB942, Berthold Technologies, Bad Wildbad, Germany) was used for the final data reading, and the experimental operation was carried out in strict accordance with the product manual.
5
Biofilm Prevention Assay for E. creticum Extracts
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A 96-well polystyrene tissue culture-treated microtiter plate was used to assess the biofilm prevention activity of E. creticum extracts. A total of 100 µL of TSB medium supplemented with 0.25% (w/v) glucose and 100 µL of extracts were added to the first well of microplates and serial twofold dilution was performed. A diluted bacterial suspension (5 µL) was added as inoculum to each well to give a final concentration of 5 × 105 CFU/mL. Wells without any plant extract were used as a positive control for biofilm formation and wells without bacterial inoculum were used as a negative control. The microplate was incubated at 37 °C for 24 h. Hereafter, the well content was removed, and the biofilm formed in the wells was fixed by heating for 1 h at 80 °C. Then, 100 µL of crystal violet (0.1%) was added to each well for staining and subsequently washed after 5 min. Finally, 100 µL of distilled water was added and the optical density (OD) was measured at 570 nm using a microplate reader (Tristar2 S LB 942, Berthold, Germany) [41 (link),42 (link)]. Minimal biofilm prevention concentration (MBPC) was defined as being the lowest concentration exhibiting the highest significant biofilm formation prevention.
6
Microvascular Permeability Assay in Mice
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ICR mice were randomly divided into four groups based on body weight. Animals were injected with 1) normal saline, 2) compound 48/80 (2.5 mg/kg as positive control), and 3) 5-HMF (7 mg/kg or 35 mg/kg as the low dose (5-HMF-L) and the high dose (5-HMF-H) groups, respectively) through the tail vein, in combination with 0.4 % Evans blue (n = 3 mice/group). Mice were euthanized (cervical dislocation after isoflurane inhalation anesthesia) 30 min after drug administration. Then the ears were removed. Each ear was dissected and placed into 800 μL acetone-saline (7:3) at 65 °C for 12 h. After centrifuging for 15 min (3000 rpm at 4 °C), 200 μL supernatants were added to a 96-well plate. Different concentrations of Evans blue solution were also added to generate a standard curve. Absorbance was measured at 620 nm on a microplate reader (Berthold, TriStar2S LB 942, Germany).
7
CellTiter-Glo Viability Assay of Gallic Acid
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The CellTiter-Glo viability test was performed according to the manufacturer’s instructions (Promega, catalogue number: G7570). Briefly, the HT29 cells (ATCC® HTB-38) were seeded in triplicate, at a 7500 cells/well concentration using 96-well opaque plates. After incubation for 24 h, the cells were treated with gallic acid and its combinations with MCM-41 or MCM-48 in concentrations ranging from 1000 µg/mL to 7.82 µg/mL for 24 h. The cells were then incubated for 10 min with CellTiter-Glo reagent, and the luminescence was measured using a 96-well microplate multi-reader Tristar2S LB942 (Berthold Technologies, Bad Wildbad, Germany). Background luminescence was measured in a cell-free medium and subtracted from experimental values.
8
Luciferase Assay for Cell Lysis
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After washing once with PBS, cells were lysed with LCβ buffer (Toyo Inki, Tokyo, Japan) at room temperature for over 30 min with intense agitation. Luciferase activity in the cleared lysates was measured by mixing a luciferin substrate (Promega, Madison, WI) using TriStar2S LB942 (Berthold Technologies, Bad Wildbad, Germany). The values of luciferase activity were standardized by the protein concentration titered by the BCA protein assay kit (Thermo Fisher Scientific).
9
Transcriptional Profiling of HDF Cells
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For each drug treatment, HDFs were seeded on a 96-well plate and grown for 24 h. The cells were transduced using each lentiviral supernatant for 48 h before treatment with NecB. Cells were then treated with NecB at the indicated concentrations for 10 days. Fluorescence was evaluated every 12 h using a multiple plate reader (TriStar2 S LB 942, Berthold technologies, Bad Wildbad, Germany), excited at 480 nm and detected at 510 nm. The light emission was normalized by that determined in blank non-treated group and the transcription factor activity was evaluated. The regulatory networks of transcription factor activation were schematized using BTNET (http://ibtnet.korea.ac.kr/ ), as described previously [66 (link)].
10
Fecal Enzyme Activity in Turkeys
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The fecal enzymes activity in the excreta of turkeys was determined after 2 days and 15 weeks of the animals rearing in seven randomly selected turkeys (three repetitions) in each experimental group. The activity of α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase and β-glucuronidase in the excreta of turkeys was determined using the spectrophotometric method using the multi-plate microplate reader TriStar2 S LB 942 (Berthold Technologies, Germany). In order to prepare samples for analysis, an ultrasonic disintegrator (Sonificator Cole-Parmer Instrument Co., USA) was used with appropriate parameters (amplitude 60 Hz, pulse 6 s, break 2 s, total time 5 min). The method is based on a color reaction between the appropriate substrate and the determined enzyme. Absorbance was measured at λ = 400 nm (α-glucosidase, α-galactosidase, β-galactosidase), λ = 450 nm (β-glucosidase) and λ = 540 nm (β-glucuronidase). The activity of the determined enzyme [μMh/g] was expressed as the amount of p-nitrophenol [μM] (α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase) or phenolphthalein (β-glucuronidase) released under specific conditions within 1 h on 1 g of the excreta.
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