All ELISAs with the exception of the hIL-18 and mIL-18 ELISA (MBL International antibodies D044-3, D045-6 and Thermofisher #BMS267-2, respectively) were from R&D Systems (#DY210, DY201, DY401, DY400, DY200). Quantification of cell death was performed by analysis of LDH release in the cell supernatant. Cell death was also quantified by monitoring propidium iodide (PI) incorporation. Briefly, 3 to 5 × 104 U937 cells, hMDMs or BMDMs were seeded in 0.3 cm2 wells of a black 96-flat-bottom-well plate. One hour after infection, PI was added at a final concentration of 5 μg.mL−1. Fluorescence was measured over a 24 h period on a microplate fluorimeter (Tecan).
Dy210
Manufactured by R&D Systems
Sourced in United States, United Kingdom, Germany
The DY210 is a laboratory instrument designed for the determination of DNA and RNA concentrations. It operates using spectrophotometric technology to measure the absorbance of samples at specific wavelengths, providing quantitative analysis of nucleic acid content.
Automatically generated - may contain errors
Lab products found in correlation
Dy206, by R&D Systems (12 mentions)
Dy201, by R&D Systems (10 mentions)
Dy401, by R&D Systems (3 mentions)
Dy217b, by R&D Systems (3 mentions)
Dy208, by R&D Systems (3 mentions)
Dy279, by R&D Systems (2 mentions)
Dy406, by R&D Systems (2 mentions)
Dy410, by R&D Systems (2 mentions)
N hydroxysuccinimide, by Thermo Fisher Scientific (2 mentions)
Albumin from bovine serum, by Merck Group (2 mentions)
Legato 210, by KD Scientific (2 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
D045 6, by R&D Systems (1 mentions)
D044 3, by R&D Systems (1 mentions)
Microplate fluorimeter, by Tecan (1 mentions)
Cobas pro, by Roche (1 mentions)
Cystatin c reagents, by Abbott (1 mentions)
Architect ci16200, by Abbott (1 mentions)
Dy240, by R&D Systems (1 mentions)
Dy285, by R&D Systems (1 mentions)
30 protocols using dy210
1
Quantification of Cell Death in Immune Cells
2
Quantifying Cytokine Release in Immune Cells
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N9 cells were seeded at the concentration of 9 × 104 cells/well into a 12-well plate and then the day after were treated as described above. Levels of TNF-α released into the culture medium were quantified after 3, 6, and 24 h by using the corresponding quantification enzyme-linked immunosorbent assay (ELISA) kits (88-7324, Thermo Fisher Scientific, Monza, Italy) according to the manufacturer’s instructions. Levels of IL-6 and IL-1β released into the culture medium were quantified after 24 h by using the corresponding quantification enzyme-linked immunosorbent assay (ELISA) kits (DY406, DY401, R&D systems, Minneapolis, MN, USA). PMA-THP-1 cells were seeded at a concentration of 8 × 104 cells/per well into a 96-well plate and differentiated as described above. Cells were then treated with 100 nM PEA or RePEA as indicated above. Levels of TNF-α, IL-6, and IL-1β released into the culture medium were quantified after 24 h by using the corresponding quantification enzyme-linked immunosorbent assay (ELISA) kits (DY210, DY206, DY201, R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The data were expressed as pg/mL following interpolation on the basis of a standard curve. The experiment was done in triplicate and repeated for three independent measurements.
3
Whole-blood cytokine production assay
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Within 30 min of blood sampling, 50 μL of whole-blood was LPS stimulated in triplicates after having been diluted 1:10 in RPMI medium (LONZA, BE12-167F) supplemented with LPS (Sigma-Aldrich, L2645-1MG) in a final concentration of 1 μg mL−1. Samples were incubated for ~24 h at 37 °C and 5% CO2 in order to determine ex vivo cytokine production. After incubation supernatants were harvested and stored at −80 °C until ELISA measurements of IL-1β, IL-6, TNF-α and IFN-γ (R&D Systems, DY201, DY206, and DY210, respectively)73 (link). The CV% was 3.6–5.2%.
4
Biomarkers for Critically Ill Patients in ICU
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Samples were taken at admission to the ICU as part of their routine procedure. Plasma creatinine [revised Lund-Malmö GFR estimating equation (LMR)] [27 (link)] was analyzed at the department of clinical chemistry and pharmacology, Uppsala University Hospital, Uppsala using an IDMS calibrated enzymatic method on Roche Cobas Pro (Roche Diagnostics, Rotkreuz, Switzerland). The laboratory is accredited by Swedac (Borås, Sweden). ISO 15189:2012 specifies the requirement for quality and competence in the laboratory, which is participating in Equalis (Uppsala, Sweden) external quality assurance programs for creatinine. Cystatin C Caucasian-Asian-Pediatric-Adult (CAPA) [28 (link)] was analyzed on an Architect ci16200 (Abbot Laboratories, Abbott Park, IL, USA) with IDMS-calibrated enzymatic creatinine reagents from the same manufacturer, and cystatin C reagents were from Gentian AS (Moss, Norway). Plasma tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) were analyzed with the commercial sandwich ELISA kits, (DY210 and DY206 R&D Systems, Minneapolis, MN, USA). The total coefficient of variations for the assays were approximately 7%.
High-sensitivity plasma CRP was analyzed by a particle-enhanced turbidimetric assay (PETIA) at ICU admission, and continuously during ICU stay. The maximal CRP was noted.
High-sensitivity plasma CRP was analyzed by a particle-enhanced turbidimetric assay (PETIA) at ICU admission, and continuously during ICU stay. The maximal CRP was noted.
5
Profiling Cytokine Levels in Biopsies
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Biopsies were pooled from five patients of each category. The homogenized sample was incubated for 5 min at room temperature to permit the complete dissociation of nucleoprotein complexes. Thereafter, the homogenate sample was centrifuged to remove cell debris and the supernatant was transferred to a new tube. The supernatant was assayed by enzyme-linked immunosorbent assay (ELISA) for IL-6, IFN-γ, TNF-α, TGF-β, IL-10, IL-8 and thymic stromal lymphopoietin (TSLP) using the manufacturer's protocol (R&D, USA; DY206, DY285, DY210, DY240, DY217B, DY1398, respectively).
6
Quantifying Cytokine Release in Co-culture
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The release of IL-1β, IL-8, TNF-α, and TGF-β1 was quantified using cell-free supernatants from the BL compartment after 4, 24, or 48 h of co-culture. The ELISA was run as described by Kinsner et al. (2006) (link) using commercially available antibody pairs (R&D Systems, Cat.: DY210, DY201, DY208, DY240; Abingdon, Oxfordshire, UK). Briefly, the primary antibodies were incubated overnight at room temperature (RT) in coating buffer (0.1 M NaHCO3 in MilliQ H2O) on high protein-binding 96-well plates (Thermo Fisher Scientific). After washing (PBS + 0.05% Tween20) and blocking (3% BSA/PBS) for 1 h at RT, the cytokine standards and supernatant samples were added and incubated for 2 h at RT, followed by incubation for 45 min with biotinylated secondary antibodies. After 30 min incubation with streptavidin-peroxidase (Biotrend Chemikalien; Cologne, NRW, Germany), 100 μL 3,3′,5,5′-Tetramethylbenzidine (Sigma-Aldrich) was added and the reaction was stopped with sulfuric acid (1 M) after 5 (IL-8) or 15 min (TNF-α, TGF-β1, and IL-1β). The absorbance was read spectrophotometrically (Enspire, Perkin Elmer; Milano, Lombardy, Italy) at 450 nm.
7
Quantifying Human TNF-α Levels
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Human TNF-α in the cell-free culture supernatant collected from transfected HEK293 or PC3 cells was measured using a commercially available enzyme-linked immunosorbent assay kit (TNF-α; R&D Systems, DY210).
8
Inflammatory Biomarkers in Plasma Characterization
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Calprotectin was measured in plasma samples with particle enhanced turbidimetric method (Gentian Diagnostics, Moss, Norway) on a Cobas c501 (Roche Diagnostics, Mannheim, Germany). Reagents, controls, and calibrators for calprotectin come from Gentian Diagnostics. Assay performances were checked using the manufacturer's control materials on two levels.
CRP was analyzed using the Cobas c501® system (Roche Diagnostics, Mannheim, Germany), using the CFAS-calibrator and Roche Modular® reagent. Assays were checked using the manufacturer's control materials on two levels. Procalcitonin, IL-6 and TNF-α were analyzed by sandwich ELISAs (DY8350, DY206, and DY210, R&D Systems, Minneapolis, MN, USA). The total coefficients of variation for the ELISA methods were approximately 6–7%. All assays were performed blinded without knowledge of clinical information.
CRP was analyzed using the Cobas c501® system (Roche Diagnostics, Mannheim, Germany), using the CFAS-calibrator and Roche Modular® reagent. Assays were checked using the manufacturer's control materials on two levels. Procalcitonin, IL-6 and TNF-α were analyzed by sandwich ELISAs (DY8350, DY206, and DY210, R&D Systems, Minneapolis, MN, USA). The total coefficients of variation for the ELISA methods were approximately 6–7%. All assays were performed blinded without knowledge of clinical information.
9
Quantifying Inflammatory Cytokines in hiPSC Cells
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Supernatants from the source of three donors or three independent batches of hiPSC-derived cells were collected, and 50 μl per well in duplicates was used for ELISA. Human IL-6 and TNFα Duoset (DY206, DY210, R&D) were carried out according to the manufacturer’s instructions. The final concentration of the cytokines was calculated with the standard curve.
10
Quantification of Cytokine Levels
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Briefly, a 96-well plate (Eppendorf, Hamburg, Germany) was coated with monoclonal Abs against TNF-α (DY210; R&D systems, Minneapolis, MN, USA), IL-17A (DY317; R&D systems), and IL-22 (MAB7822, 782-IL, BAM7821; R&D systems) at 4°C overnight. After blocking with PBS/1% bovine serum albumin/0.05% Tween 20 for 2 h at room temperature (22°C–25°C), the test samples and recombinant TNF-α, IL-17A, and IL-22 as standards were added to the 96-well plate and incubated at room temperature for another 2 h. The plates were washed 4 times with PBS and Tween 20, and then incubated with biotinylated human monoclonal Abs against TNF-α, IL-17A, and IL-22 for 2 h at room temperature. After washing, the streptavidin–alkaline phosphate–horseradish peroxidase conjugate (Sigma, St. Louis, MA, USA) was added to the wells for 30 min, and incubation with 1 mg/ml p-nitrophenyl phosphate (Sigma) dissolved in diethanolamine (Sigma) to develop the color reaction. The reaction was stopped by the addition of 1 M NaOH, and the optical density of each well was measured at 405 nm.
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