The quantification of interleukin IL-17A and TNF-α levels in serum in the defined rat groups were determined quantitatively using commercially available ELISA kits (Invitrogen, ThermoFisher Scientific). Analytical procedures were performed according to the manufacturer’s instructions. All measurements were made in duplicates. According to the standards, readouts and calculations of concentrations were performed using a microplate reader (Bio-Tek EL808, Winooski, VT, USA) and the Bio-Tek Instruments Inc. software, EL808. Results are expressed as pg/mL of serum (mean ± SD).
El808
Manufactured by Agilent Technologies
Sourced in United States, United Kingdom
The EL808 is a laboratory equipment product offered by Agilent Technologies. It is a microplate reader device designed for various analytical applications. The core function of the EL808 is to measure the absorbance, fluorescence, or luminescence of samples in microplates.
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Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (7 mentions)
96 well plate, by Corning (6 mentions)
Mts tetrazolium reagent, by Promega (6 mentions)
Nunc maxisorp plate, by Thermo Fisher Scientific (5 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (5 mentions)
Celltiter 96 aqueous assay, by Promega (4 mentions)
Automatic handheld cell counter, by Merck Group (3 mentions)
Elx405select cw, by Agilent Technologies (3 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (3 mentions)
Bca method, by Elabscience (2 mentions)
Gen5 data analysis software, by Agilent Technologies (2 mentions)
Menadione, by Merck Group (2 mentions)
D4522, by Merck Group (2 mentions)
Cck 8 kit, by Dojindo Laboratories (2 mentions)
Sirius red f3b dye, by Avantor (2 mentions)
Bovine serum albumin (bsa), by Biosynth (2 mentions)
Cetylpyridinium chloride, by Merck Group (1 mentions)
Tnf α elisa kit, by BioLegend (1 mentions)
P0260, by Agilent Technologies (1 mentions)
Deoxynivalenol, by Merck Group (1 mentions)
106 protocols using el808
1
Quantification of Serum IL-17A and TNF-α
2
Optimal hADSC-Exosomes for Osteogenesis
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To determine the optimal time-span of extracted hADSC-Exos during hADSCs osteogenic differentiation, the hADSCs of passage 4 were seeded into the 24-well plates, and was cultured in PM. After getting 70–80% confluence, the culture medium was replaced by PM containing exosomes (Exo0d, Exo1−14d and Exo15−28d) from different time-span of hADSCs osteogenesis induction. hADSCs cultured in exosomes-free PM and OM were set as negative and positive controls, respectively.
The mineralized deposits were evaluated by ARS after incubation for 21 days. After fixation by 4% paraformaldehyde for 30 min, the cells were washed three times with PBS before staining with Alizarin red S for 5 min, and then the samples were washed again with PBS. For ARS quantitation the mineralized deposits were dissolved in 10% cetylpyridinium chloride (Sigma, USA) in dark for 30 min at room temperature. The solution was transferred to a 96-well plate with 100 μL/well. The optical density (OD) values was measured at 562 nm using a microplate reader (BioTek EL808, USA).
The mineralized deposits were evaluated by ARS after incubation for 21 days. After fixation by 4% paraformaldehyde for 30 min, the cells were washed three times with PBS before staining with Alizarin red S for 5 min, and then the samples were washed again with PBS. For ARS quantitation the mineralized deposits were dissolved in 10% cetylpyridinium chloride (Sigma, USA) in dark for 30 min at room temperature. The solution was transferred to a 96-well plate with 100 μL/well. The optical density (OD) values was measured at 562 nm using a microplate reader (BioTek EL808, USA).
3
SARS-CoV-2 S Antigen Binding Assay
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Purified S antigens (1 µg/ml) or bi-, tetra-, and hexa-valent HCAbs (37.5 nM) were coated onto 96-well NUNC Maxisorp plates (Thermo Fisher Scientific) at 4°C overnight, followed by three washing steps with phosphate-buffered saline (PBS) containing 0.05% Tween 20. Plates were blocked with blocking buffer (PBS containing 3% bovine serum albumin [BSA; Fitzgerald, Acton, MA, USA] and 0.1% Tween 20) at room temperature (RT) for 2 hours. HCAbs or S antigens were allowed to bind to the plates at fivefold or fourfold serial dilutions, starting at 5 µg/ml (HCAbs) or 0.975 mM (S antigens) diluted in blocking buffer at RT for 1 hour. HCAb binding to the S proteins was determined using a 1:2000 diluted HRP-conjugated goat anti-human IgG (ITK Southern Biotech, Uden, NL) for 1 hour at RT. S antigen binding to HCAbs was determined using a 1:4000 diluted StrepMAB-Classic HRP (IBA). HRP activity was measured at 450 nm using tetramethylbenzidine substrate (BioFX, Eden Prairie, MN, USA) and an ELISA plate reader (EL-808, BioTek, Bornem, Belgium).
4
Cell Viability Quantification Assay
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Cell viability was determined by using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). Briefly, cells (10,000 cells/well) were seeded into 96-well plates (Corning Incorporated, Corning, NY, USA) and then treated. Next, cells were incubated with 20 µL of CellTiter, and the absorbance at 490 nm was recorded using a microplate reader (EL-808, BIO-TEK Instruments Inc., Winooski, VT, USA). IC50 was evaluated by GraphPad Prism 5.4.2 Software (GraphPad Software, Boston, MA, USA).
5
Quantifying TNF-α and sEH Activity in Rat Brain
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Rat TNF-α levels in brain cortex tissue were measured by ELISA kits (TNF-α ELISA kit [B167329, Biolegend, San Diego, CA]) according to the manufacturers’ protocols. Activity of sEH in rat brain cortex was measured by incubating brain homogenate with 14,15-EET for 1 h and assaying for 14,15-DHET with an ELISA kit (DH1, Detroit R&D, Detroit, MI, USA). The optical density values were read on a plate reader (EL808, BioTek Instruments, Winooski, VT, USA) and normalized for protein concentration.
6
ACE2 Binding Assay for Coronavirus S1 Proteins
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The ability of the HCoV-NL63 S1-mFc and S1-B-mFc chimeric proteins to bind the ACE2-hFc receptor was evaluated with an ELISA-based assay. 100 μl of hACE2-hFc (20 μg/ml, diluted in PBS) was coated on a 96-well MaxiSorb plate overnight at 4 °C. Nonspecific binding sites were subsequently blocked with a 3% (w/v) solution of bovine serum albumin in PBS. Plates were washed with washing buffer (PBS with 0.05% Tween 20) and subsequently incubated with serially diluted S1-mFc proteins (starting with equimolar concentrations) for 1 h at room temperature, after which plates were washed three times with washing buffer. mFc-tagged S1 proteins were detected with HRP-conjugated polyclonal rabbit-anti-mouse immunoglobulins (1:2,000 dilution in PBS with 0.1% BSA; DAKO, P0260), and a colorimetric reaction was produced after incubation with tetramethylbenzidine substrate (BioFX). The optical density (OD) was subsequently measured at 450 nm with an ELISA reader (EL-808, BioTEK). Background (signal from HRP-conjugated anti-mFc antibody alone) was subtracted from the OD450nm values. The mFc-tagged SARS-CoV S1 subunit was used as a positive control, whereas the mFc-tagged HCoV-NL63 S1 domain 0 (HCoV-NL63 S1-0-mFc) and PEDV S1 subunit (PEDV S1-mFc), both of which do not bind ACE2, were used as negative controls.
7
Quantifying Inflammatory Markers in Brain Tissue
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COX-2, p-NF-κB, and TNF-α expression were quantified using rat ELISA kits following the manufacturer's instructions. Briefly, an appropriate quantity of brain tissue (50 mg) was homogenized using a Heidolph crusher at 15,000 rpm in 2500 μL PBS containing PMSF as the protease inhibitor [56 (link)]. Next, the tissue homogenate was centrifuged at 4000×g for 10 min, and the supernatant was collected. Total protein concentration in the supernatant of each group was calculated using the BCA method (Elabscience); moreover, an equivalent protein quantity was used to quantify the protein concentration of COX-2, p-NF-κB, and TNF-α using an ELISA microplate reader (BioTek EL×808). Finally, the protein concentration (pg/mL) was normalized to the total protein content (pg/mg total protein).
8
Mycotoxin Detection Protocol
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T-2 toxin, HT-2 toxin, ochratoxin A, ochratoxin B, aflatoxin B1, deoxynivalenol, fumonisin B1, zearalenone, methanol, hexane, dichloromethane, phosphoric acid, sulphuric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium carbonate, sodium bicarbonate, sodium chloride, Tween 20, bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), horseradish peroxidase (HRP), gelatine, dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Dorset, UK and Zwijndrecht, The Netherlands). 3,3′,5,5′-Tetramethylbenzidine (TMB) was obtained from Neogen (Lansing, MI, USA).
A laboratory mill IKA A11 Basic was used for sample blending. Sigma 4K10 centrifuge was used for samples centrifugation and BioTek EL808 type ELISA plate reader for reading the microtiter plates.
A laboratory mill IKA A11 Basic was used for sample blending. Sigma 4K10 centrifuge was used for samples centrifugation and BioTek EL808 type ELISA plate reader for reading the microtiter plates.
9
ELISA Protocol for Antibody Binding
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NUNC Maxisorp plates (Thermo Scientific) were coated with equimolar antigen amounts at 4 °C overnight. Plates were washed three times with PBS containing 0.05% Tween-20 and blocked with 3% bovine serum albumin (Bio-Connect) in PBS containing 0.1% Tween-20 at room temperature for 2 hours. Fourfolds serial dilutions of mAbs starting at 10 µg/ml (diluted in blocking buffer) were added and plates were incubated for 1 hour at room temperature. Plates were washed three times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human secondary antibody (ITK Southern Biotech) diluted 1:2000 in blocking buffer for 1 hour at room temperature. An HRP-conjugated anti-StrepMAb (IBA, Catalog# 2-1509-001) antibody was used to corroborate equimolar coating of the strep-tagged spike antigens. HRP activity was measured at 450 nanometer using tetramethylbenzidine substrate (BioFX) and an ELISA plate reader (EL-808, Biotek). Half-maximum effective concentration (EC50) binding values were calculated by non-linear regression analysis on the binding curves using GraphPad Prism (version 8).
10
Time-resolved Growth Assay of S. aureus
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The time-resolved growth analysis was performed in 96-well microtiter plates by incubating the antibacterial agent with Staphylococcus aureus ATCC 25923. Briefly, exponentially grown S. aureus ATCC 25923 (OD600 = 0.3–0.5) was diluted to a final concentration of 5 × 104 CFU/mL in MHB. Then, 50 µL of this culture was added to the wells of the plate containing the MIC or ½ MIC of the compound to be tested. The plates were incubated overnight (24 h) at 37 °C in the microplate reader (Bio-Tek (Winooski, VT, USA) EL808) with shaking, recording the OD600 at 10 min intervals. The results obtained were compared to the untreated control and represent the mean of two independent experiments performed in triplicate.
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