The anti-inflammatory activity of the extracts (400 to 6.25 μg/mL) was evaluated based on nitric oxide (NO) production in a RAW 264.7 murine macrophage cell line (ECACC 91062702) due to lipopolysaccharide (LPS, 1 mg/mL in DMEM; Sigma-Aldrich, Saint Louis, MO, USA) stimulation. For that purpose, the Griess Reagent System kit (Promega, Madison, WI, USA) was used as described in previously published protocols [32 (link)]. Dexamethasone (50 mM) (Sigma-Aldrich, Saint Louis, MO, USA) and samples without the addition of LPS were used as positive and negative controls, respectively. The NO generated was monitored at 540 nm (ELX800 Biotek microplate reader; Bio-Tek Instruments Inc., Winooski, VT, USA). The extract concentrations that trigger 50% of NO production inhibition were expressed as EC50 values (μg/mL).
Elx800 biotek microplate reader
Manufactured by Agilent Technologies
Sourced in United States
The ELX800 Biotek microplate reader is a compact and versatile instrument designed for measuring absorbance in microplates. It features a monochromator-based optical system that provides accurate and reliable results across a wide range of wavelengths. The ELX800 supports multiple microplate formats and can perform a variety of plate-reading applications, such as endpoint, kinetic, and spectral scanning.
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Lab products found in correlation
Griess reagent system kit, by Promega (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Cck 8 kit, by Beyotime (1 mentions)
Nuclear extraction kit, by Abcam (1 mentions)
Bca protein quantification kit, by Abcam (1 mentions)
Serum separator tube, by BD (1 mentions)
Microcentrifuge tube, by Eppendorf (1 mentions)
Enzyme linked immunosorbent assay, by ALPCO (1 mentions)
8 protocols using elx800 biotek microplate reader
1
Anti-inflammatory Activity of Extracts via NO Assay
2
Cell Viability Assay Protocol
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Viability of the transfected cells was determined using a CCK-8 kit (Beyotime Institute of Biotechnology). In brief, after transfection had been allowed to proceed for 24 and 48 h, 10 µl CCK-8 reagents were added into the transfected cells and co-cultured at 37°C under an atmosphere of 5% CO2 for 2 h. The optical density (OD) values of the cells were measured using an ELX800 BioTek microplate reader (Biotek Instruments, Inc.) at 450 nm, and the OD values of the cells was measured at 24, 48, 72 and 96 h, respectively.
3
Minocycline Cytotoxicity and Protective Effects
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Human C28/I2 chondrocytes were cultured in T25 flasks and trypsinized after reaching 80 % confluence. Trypsinized cells were centrifuged at 1000 rpm and seeded (5x103/well) in 96-well plates. First, cells were treated with minocycline (5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 µM) for 24, 48, and 72 hours at 37 °C to determine its cytotoxicity in a time-dependent manner. Second, to detect the inhibitory concentration (IC50) and effective concentration (EC50) of minocycline, cells were treated with minocycline with various concentrations for 24 h at 37 °C. Untreated cells were used as the control group. Third, cells were pre-treated with various concentrations of minocycline for 2 hours before adding IL-1β (10 ng/ml) to determine the protective effect of minocycline against IL-1β cytotoxicity. So, each of the four groups (A, B, C, D) of the cells was treated with 50 μl MTT (2 mg/ml) for 3 h at 37 °C. Purple formazan crystals were dissolved using DMSO (100 μl/well) after incubation. An ELx 800 BioTek microplate reader (San Francisco, CA, USA) was used to measure the absorbance at 570 nm.
4
Nrf2 Activation in HepG2 Cells
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A total of 1×10
6 HepG2 cells/well were cultured in DMEM. The medium was discarded, and the cells were exposed to the extract (0.25 or 1 µg/mL), compounds or controls (0.5 or 2 µM) for 0, 4, 12 or 24 hours. After exposure, cells were harvested and used for the simultaneous extraction of nuclear and cytosolic proteins following the specifications included in the Nuclear Extraction Kit (Abcam, Cambridge, UK, ab113474). Total protein was quantified using the BCA Protein Quantification Kit (Abcam, Cambridge, UK, ab102536). Nrf2 was detected by using the Nrf2 Transcription Factor Assay Kit (Colorimetric, Abcam, Cambridge, UK, ab207223) following the manufacturer’s instructions. The absorbance of each well was measured at 450 nm in an ELx800 BioTek microplate reader.
6 HepG2 cells/well were cultured in DMEM. The medium was discarded, and the cells were exposed to the extract (0.25 or 1 µg/mL), compounds or controls (0.5 or 2 µM) for 0, 4, 12 or 24 hours. After exposure, cells were harvested and used for the simultaneous extraction of nuclear and cytosolic proteins following the specifications included in the Nuclear Extraction Kit (Abcam, Cambridge, UK, ab113474). Total protein was quantified using the BCA Protein Quantification Kit (Abcam, Cambridge, UK, ab102536). Nrf2 was detected by using the Nrf2 Transcription Factor Assay Kit (Colorimetric, Abcam, Cambridge, UK, ab207223) following the manufacturer’s instructions. The absorbance of each well was measured at 450 nm in an ELx800 BioTek microplate reader.
5
Anti-inflammatory Activity in Murine Macrophages
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The anti-inflammatory activity of the extracts was measured within a range of concentrations from 400 to 6.25 μg/mL based on nitric oxide (NO) production by a lipopolysaccharide (LPS)-stimulated murine macrophage cell line (RAW 264.7). NO production was quantified based on the nitrite concentration utilizing the Griess Reagent System kit, which contains sulphanilamide, N-1-naphthylethylenediamine dihydrochloride, and nitrite solutions, based on previously published protocols [48 (link)]. Dexamethasone (50 mM) was used as a positive control and samples without LPS served as the negative control.
The nitric oxide generated was defined by reading absorbances at 540 nm (ELX800 Biotek microplate reader, Bio-Tek Instruments, Inc., Winooski, VT, USA) and by contrast with the standard calibration curve. Results were expressed as EC50 values (µg/mL), representing the extract concentration that triggers 50% of NO production inhibition.
The nitric oxide generated was defined by reading absorbances at 540 nm (ELX800 Biotek microplate reader, Bio-Tek Instruments, Inc., Winooski, VT, USA) and by contrast with the standard calibration curve. Results were expressed as EC50 values (µg/mL), representing the extract concentration that triggers 50% of NO production inhibition.
6
MTT Assay for Cell Viability
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After 24 h of incubation with different MeCc concentrations, the media were discarded, and adherent cells were incubated with 100 µl/well MTT at a concentration of 0.5 mg/ml prepared in PBS and subsequently incubated at 37 °C for additional 3 h at 37 °C under dark condition (Aboul-Soud et al., 2020 , Mosmann, 1983 (link)). Then, 100 μl isopropyl alcohol was added per well in order to dissolve the purple formazan crystals by the help of shaking for another 2 h at room temperature. Subsequently, the absorbance was measured at 549 nm using ELX 800 BioTek microplate reader (BioTek Instruments, Winooski, VT, USA). The results were analyzed in triplicates and the viability percentage were calculated. The MeCc concentrations resulting in 50% growth inhibition of HepG2 cells (IC50) were calculated by use of Microsoft Excel trendline equation (Aboul-Soud et al., 2020 ).
7
Serum CRP Measurement Protocol
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Participants were instructed to avoid strenuous physical exercise for 48 hours before each blood draw and completed a 24-h diet recall prior to blood collection in visit 1 in which they were asked to repeat prior to blood collection in visit 5. Whole blood was collected into serum separator tubes (Beckton Dickinson, East Rutherford, NJ, USA) by a certified phlebotomist, allowed to clot for 30-min at room temperature, then centrifuged at 2000 RPM for 15 min. The serum was pipetted into 1.5 mL microcentrifuge tubes (Eppendorf AG, Hamburg, Germany) and immediately stored in a −80 °C freezer. Serum concentrations of CRP were determined with a commercially available enzyme-linked immunosorbent assay (ALPCO Diagnostics, Salem, NH, USA). Microplates were read with an ELx800 BioTek microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at the recommended wavelength of 450 nanometers.
8
Cholinesterase Inhibitory Assay Protocol
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AChE and BuChE-Inhibitory activities were assayed as described by Ellman et al. (1961) (link) with some modifications by Ortiz et al. (2016) (link). 50 μL of AChE in buffer phosphate (8 mM K2HPO4, 2.3 mM NaH2PO4, 0.15 M NaCl, 0.05% Tween 20, pH 7.6) and 50 μL of the sample dissolved in the same buffer were added to the wells. The plates were incubated for 30 min at room temperature before the addition of 100 μL of the substrate solution (acetylthiocholine or butyrylthiocholine, 0.1 M Na2HPO4, 0.5 M DTNB, 0.6 mM ATCI in Millipore water, pH 7.5). The absorbance was read in a ELx800 Biotek microplate reader (Vermont, USA) at 405 nm after three minutes. Enzyme activity was calculated as a percentage compared to an assay using a buffer without any inhibitor. The inhibitory data were analyzed with the software Prism (Graph Pad Inc., San Diego, CA, USA). IC50 values are means ± SD of three individual determinations each performed in triplicate.
The enzymes used for the test were AChE of the electric eel Electrophorus electricus (C3389) and Equine serum BuChE (C7512), while the substrates in each case were acetylthiocholine iodide (A5751) and butyrylthiocholine iodide (20820). Dithionitrobenzoic acid (DTNB, D-8130) was added to generate the color reaction. Galanthamine was the reference compound. All the reagents of this assay were purchased from Sigma-Aldrich (Missouri, USA).
The enzymes used for the test were AChE of the electric eel Electrophorus electricus (C3389) and Equine serum BuChE (C7512), while the substrates in each case were acetylthiocholine iodide (A5751) and butyrylthiocholine iodide (20820). Dithionitrobenzoic acid (DTNB, D-8130) was added to generate the color reaction. Galanthamine was the reference compound. All the reagents of this assay were purchased from Sigma-Aldrich (Missouri, USA).
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