To measure the cytokine production of MLN cells, MLNs were removed from mice, in non-infected mice and at days 1, 3, 5 and 7 after viral infection. MLN cells were plated at 2.5 × 105 cells/ml in a 96 well U-bottom plate stimulated with plate-bound 1 μg/ml anti-CD3 antibody (clone: 2C11) in DMEM containing 10% FBS. After culturing for 48 h, the supernatants were collected to measure the IFN-γ and IL-4 levels by ELISA methods (R&D systems, MN, USA).
Elisa method
Manufactured by R&D Systems
Sourced in United States
The ELISA method is a laboratory technique used for the detection and quantification of specific proteins or other molecules in a sample. It utilizes enzyme-linked immunosorbent assay technology to measure the concentration of the target analyte. The core function of the ELISA method is to provide a sensitive and reliable method for measuring the levels of a specific substance in a complex mixture.
Automatically generated - may contain errors
Lab products found in correlation
El800, by Agilent Technologies (2 mentions)
Beckman automatic biochemical apparatus, by Beckman Coulter (2 mentions)
Mtt based in vitro toxicology assay kit, by Merck Group (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Vitros 250, by Johnson & Johnson (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Sandwich elisa method, by BioVendor (1 mentions)
Multiskan ascent, by Thermo Fisher Scientific (1 mentions)
Facscalibur system, by Beckman Coulter (1 mentions)
Vivaspin 500, by Sartorius (1 mentions)
32 protocols using elisa method
1
Cytokine Production in Viral Infection
2
Quantification of Cytokines and MMPs
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After 24 h of incubation, the supernatant was collected and stored at −80 °C. To quantify cytokines, the human cytometric bead array Th1/Th2/Th17A kit (Becton, Dickinson and Company, San Jose, California, USA) was used to detect interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 17A (IL-17A), Tumour Necrosis Factor alpha (TNF-α) and Interferon Gamma (IFN-γ). This was performed on a BD FACSVerse flow cytometer, and analysed using BD FCAP array software. Measurement of IL-1β and IL-12 were performed by ELISA as they are not included in the CBA kit. MMP-1, -2, -8, and -9 were also quantified using ELISA assays. Information was also collected on history of tuberculosis (TB) and having a BCG scar, while HIV testing was done on all participants. Cell viability (results not shown) was assessed using the MTT-based in vitro toxicology assay kit (Sigma, Dorset, UK) which confirmed viability of the tissue during incubation. The results were exported into Stata 14 software (College Station, Texas, USA) for analysis. A subset of 13 samples chosen at random were used to quantify levels of MMP-1, -2, -8 and -9 secretion using ELISA methods (R&D Systems, Abingdon, UK).
3
Analyzing IL-18 Mutant Effects on NK-92MI Cells
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The NK-92MI cells were maintained in complete α-MEM medium supplemented with 12.5% FBS and 12.5% horse serum at 37°C in 5% CO2 humidified air. For the assays, NK-92MI cells were suspended at 0.5 × 106 cells per ml in complete α-MEM medium and stimulated in 0.2-ml volumes in 96-well plates with 0.5 ng/ml of IL-12 and different concentrations (200, 100, 50, 25 and 12.5 ng mL-1) of recombinant IL-18, or the five mutants. After 16–20 h at 37°C in humidified air with 5% CO2, the culture supernatants were collected for IFN-γ measurement by the ELISA method (R&D system).
4
Cytokine and Autoantibody Profiles in JIA
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Additionally, serum and SF concentrations of IL-1β, IL-6, IL-17, IL-21, IL-23, and TNF-α in both JIA children and the controls were evaluated by ELISA method (R&D; Minneapolis, MN, USA and BioVendor; Brno, Czech Republic, respectively).
Finally, in all JIA children particular laboratory parameters were measured, such as blood morphology, including white blood cell (WBC) and platelet (PLT) counts, erythrocyte sedimentation ratio (ESR), serum C-reactive protein (CRP) levels, rheumatoid factor (RF), antinuclear antibodies (ANA), anti-cyclic citrullinated peptide (anti-CCP) antibodies (Table 1 ), all by routine methods.
Finally, in all JIA children particular laboratory parameters were measured, such as blood morphology, including white blood cell (WBC) and platelet (PLT) counts, erythrocyte sedimentation ratio (ESR), serum C-reactive protein (CRP) levels, rheumatoid factor (RF), antinuclear antibodies (ANA), anti-cyclic citrullinated peptide (anti-CCP) antibodies (
5
Comprehensive biomarker assessment protocol
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Fasting blood samples were drawn in the morning from the antecubital vein using minimal stasis. Lipid profile, glucose, liver enzymes, urine, creatinine with eGFR, complete blood cell and platelet count were analyzed by routine laboratory techniques. C-reactive protein (CRP) was measured using analyzer VITROS 250 Johnson & Johnson. Blood samples were drawn into serum separation tubes, centrifuged at 2000×g for 20 min at room temperature, within 2 h from sampling. The supernatant was frozen in aliquots and stored at -70 °C until analysis. Interleukin-6 (IL-6), VCAM-1, and soluble thrombomodulin were measured using standardized ELISA method (all, R&D Systems, Minneapolis, MN, USA). Anti-PR3 IgG was measured in all GPA subjects using ELISA assay (EUROIMMUN, Lübeck, Germany).
6
Measuring Airway Inflammation Biomarkers
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Mouse elastase and total proteins in air pouch exudates were measured using respectively an ELISA method (R&D Systems, Minneapolis, MN, USA) and a protein assay (Bio-Rad, Mississauga, ON, Canada) per the manufacturers' instructions.
7
Cytokine Profile Quantification by ELISA
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Culture supernatants were collected at 72 h, and the levels of IFN-γ, TNF-α, IL-12, IL-4, and IL-10 were titrated in culture supernatants using the ELISA method (R&D Systems, USA). Briefly, the plates were coated with anti-IFN-γ, anti-TNF-α, anti-IL-12, anti-IL-4, and anti-IL-10 mAb in PBS, pH 7.4, and incubated at 4 °C overnight. After blocking the wells using buffer containing PBS plus 0.05% (v/v) Tween 20 and 0.1% (w/v) BSA, supernatants were added to each well. Biotin-labeled mAb in incubation buffer was added to each well, and streptavidin-HRP was used as enzyme. The reaction was developed using 3,3’,5,5’-tetramethylbenzidine substrate and stopped with 2.5 M H2SO4 solution. The plates were washed after each step of incubation using PBS plus 0.05% (v/v) Tween 20. Minimum sensitivity levels were 63pg/mL for IFN-γ, 63pg/mL for TNF-α, 62pg/mL for IL-12, 78pg/mL for IL-4, and 78pg/mL for IL-10. All experiments were performed using 96-well plates (COSTAR, Corning Inc., USA) and according to the instructions of R&D Systems. The reading was performed using a microplate automatic reader (EL800; Biotek, Winooski, VT, USA) at a wavelength of 450 nm.
8
Bronchoalveolar Lavage Fluid Extraction
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The bronchoalveolar lavage fluid was taken by a method as described by Han et al. [16 (link)]. Briefly, the left lungs were excised integrally from rats in each group, the bronchoalveolar lavage (BAL) was made with intratracheal injections of 2 mL of physiological saline at 37°C three times. The BALF was retrieved and centrifuged, and TNF-α and IL-8 were then determined by the ELISA method (R&D Systems Inc., Minneapolis, MN, USA).
9
Biomarkers of Inflammatory Response
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High-sensitivity C-reactive protein (hs-CRP) concentration in the serum was determined by the latex immunoturbidimetric method (BioSystems, Spain) and expressed in mg/l. The inter- and intra-assay coefficients of variations (CV) were 2.3% and 5.5%, respectively.
The serum ceruloplasmin (CER) oxidase activity was measured using the p-phenylenediamine kinetic method by Richterich [19 ] and expressed in mg/dl after a calibration with pure ceruloplasmin isolated from a healthy donor serum pool. The inter- and intra-assay coefficients of variations (CV) were 3.1% and 6.1%, respectively.
The plasma interleukin 6 (IL-6) and soluble intercellular adhesion molecule-1 (sICAM-1) concentrations were determined using the ELISA method from R&D Systems (USA). The concentrations of IL-6 and sICAM-1 were expressed in pg/ml and ng/ml. The inter- and intra-assay coefficients of variations (CV) were 5.1% and 8.8%, respectively, for IL-6 and 4.8% and 9.1%, respectively, for sICAM-1.
The serum ceruloplasmin (CER) oxidase activity was measured using the p-phenylenediamine kinetic method by Richterich [19 ] and expressed in mg/dl after a calibration with pure ceruloplasmin isolated from a healthy donor serum pool. The inter- and intra-assay coefficients of variations (CV) were 3.1% and 6.1%, respectively.
The plasma interleukin 6 (IL-6) and soluble intercellular adhesion molecule-1 (sICAM-1) concentrations were determined using the ELISA method from R&D Systems (USA). The concentrations of IL-6 and sICAM-1 were expressed in pg/ml and ng/ml. The inter- and intra-assay coefficients of variations (CV) were 5.1% and 8.8%, respectively, for IL-6 and 4.8% and 9.1%, respectively, for sICAM-1.
10
Adipose-Derived Biomarker Evaluation
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The group of circulating biomarkers produced by adipose tissue only (adiponectin, leptin receptor, leptin, and FABP‐4) and by both adipose tissue and the liver (RBP‐4 and fetuin‐A) were evaluated. Blood samples were collected after a minimum of 8 hours of overnight fasting and analyzed following standard protocols. Plasma levels of adiponectin, leptin receptor, leptin, and RBP‐4 levels were determined by the ELISA method (R&D Systems, Minneapolis, MN) with mean interassay coefficients of variation of 2.23% for adiponectin, 4.01% for leptin receptor, 4.97% for leptin, and 2.18% for RBP‐4. Plasma levels of FABP‐4 and fetuin‐A were assessed by the sandwich ELISA method (BioVendor Research and Diagnostic Products, Candler, NC) with mean interassay coefficients of variation of 2.38% for FABP‐4 and 2.52% for fetuin‐A.
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