Sandwich Enzyme-Linked Immunosorbent assay (ELISA) kits were performed to detect the concentrations for IL-1β (BioLegend’s ELISA MAXTM Deluxe Set, cat n° 432604), IL-6 (BioLegend’s ELISA MAXTM Deluxe Set, cat n° 431304), TNF-α (BioLegend’s ELISA MAXTM Deluxe Set, cat n° 430904), and IL-10 (Thermo Fisher, cat n° 88-7105-88). The protocols were performed according to the manufacturer’s instructions. An automated microplate reader (Varioskan® Flash-Thermo Fisher Scientific, Vantaa, Finland) at 450 nm was utilised to read the absorbance. The absorbance at 570 nm was subtracted from the absorbance at 450 nm to eliminate contaminations. The concentrations were calculated based on the standard curve run on each assay plate. Two independent experiments were conducted and subjected to statistical analysis. Since both experiments yielded statistical differences, the data from one of them are presented herein.
Elisa maxtm deluxe set
Manufactured by BioLegend
Sourced in United States
The ELISA MAXTM Deluxe Set is a laboratory equipment product designed for performing enzyme-linked immunosorbent assay (ELISA) experiments. The set includes the necessary components to conduct ELISA tests, but no further details on its intended use or functionality are provided.
Automatically generated - may contain errors
Lab products found in correlation
Multiskan go, by Thermo Fisher Scientific (2 mentions)
Maxisorp microplate, by Thermo Fisher Scientific (1 mentions)
680 microplate reader, by Bio-Rad (1 mentions)
Varioskan flash, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Gene5, by Agilent Technologies (1 mentions)
Hematoxylin and eosin, by Merck Group (1 mentions)
Il 13, by Novus Biologicals (1 mentions)
Masson s trichrome stain, by Merck Group (1 mentions)
Alpha naphthylisothiocyanate anit, by Merck Group (1 mentions)
Il 10, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
16 protocols using elisa maxtm deluxe set
1
Cytokine Quantification via Sandwich ELISA
2
Cytokine Levels in Colitis Model
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The concentration levels of the pro-inflammatory (TNF-α) and anti-inflammatory (IL-10) cytokines in the colonic homogenates of non-colitis, colitis, as well as the three treatment mice groups was determined by sandwich ELISA. The colon tissues (middle region) of experimental animals were collected from liquid nitrogen storage and allowed to thaw. The thawed tissues were gently teased with sterile needles and the contents were centrifuged at 2,000 × g for 30 min. The supernatant, i.e., colonic extract, was collected and analyzed for the aforementioned cytokines using the commercial kit (BioLegend’s ELISA MAXTM Deluxe Set) following the manufacturer’s protocol.
3
Quantification of IL-1β and TNF-α
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The concentrations of IL-1β and TNF-α in the culture supernatant were quantified by a sandwich enzyme-linked immunosorbent assay (ELISA) using ELISA MAXTMDeluxe Set (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions.
4
ELISA Quantification of Cytokines
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The levels of IL‐6 and TNF‐α in the culture medium of the treated cells were determined by sandwich Enzyme Link Immuno‐Sorbent Assay (BioLegend's ELISA MAXTM Deluxe Set, CA). Briefly, 100 μL of supernatant after treatment was used and assayed according the manufacturer's protocol for the relevant ELISA kit.
5
Quantification of Inflammatory Cytokines
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Cell culture supernatants were collected and stored at −70 °C until analysis. Concentration of IL-1β, IL-6, IL-10, and TNF-α was measured using the sandwich-type immunoassay ELISA MAXTM Deluxe Set (BioLegend, San Diego, CA, USA) for each molecule. Briefly, 96-well MAXISORP microplates (ThermoFisher Scientific, Waltham, MA, USA) were coated with anti-human IL-1β, anti-human IL-6, anti-human IL-10, or anti-human TNF-α capture antibody, and then blocked. Diluted supernatants were added (1–2 h, RT). After four washes, each protein was recognized with its specific biotinylated detection antibody. Then, avidin-HRP and tetramethylbenzidine (TMB) were added. Reactions were read with a microplate reader spectrophotometer (MULTISKAN GO, ThermoScientific, Waltham, MA, USA).
6
Measuring IL-6 in Cell Culture
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According to the manufacturer’s protocol, the human IL-6 ELISA MAXTM Deluxe Set (BioLegend, CA, USA) was used to measure the IL-6 level in the cell culture supernatant. A microplate reader was used to measure the absorbance at 450 nm. The data are reported in pg/mL protein and are presented as the means ± standard errors of the means (S.E.M.) of 5 independent samples.
7
Modulating Cytokine Induction by L. fermentum
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To examine whether pre-treatment of L. fermentum KGC1601-cultured media and the isolated EPS decrease the induction of cytokines by LPS, RAW 264.7 cells were seeded at the density of 7.5 × 105 cells/well in a 12-well plate and incubated for 24 h. Each sample was pre-treated with the cells for 18 h; L. fermentum KGC1601-cultured media were treated with concentrations ranging from 250 to 1000 μg/mL, and EPS was treated with concentrations ranging from 100 to 1000 μg/mL. LPS was then added to the medium at the concentration of 1 mg/mL to induce inflammation. The cells were incubated for 18 h under this condition. The culture medium obtained from each well was centrifuged at 10,000 rpm for 3 min, and the supernatants were collected. Cytokines were quantified by ELISA MAXTM Deluxe Set (BioLegend, SanDiego, CA, USA) according to the manufacturer’s recommendations. The cytokines in the culture medium were quantified as described above.
8
Myocardial IL-10 Quantification
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Heart homogenates were prepared and IL-10 concentration in myocardium was evaluated with ELISA kit (BioLegend, ELISA MAXTM Deluxe Set#431414).
9
Quantifying Macrophage Cytokine Response
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At the end of the treatment and stimulation period (day 7), macrophages were stimulated with A. fumigatus at an MOI of 1 for 24 h. Culture supernatants were harvested and analyzed for cytokines including IL-1ß, TNFα, IL-10, IL-8, IL-15, and IL-6, using commercially available sandwich enzyme-linked immunosorbent assay kit (ELISA MAXTM Deluxe Set, Biolegend, San Diego, CA, USA) specific for each cytokine as per manufacturer’s instructions. Absorbance was determined using the Biorad 680 microplate reader at 450 nm/570 nm filters (Biorad, Hercules, CA, USA).
10
Serum IL-22 and hsCRP Measurement Protocol
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Following a 12-hour overnight fast, blood samples were drawn from the participants’ antecubital vein. Serum samples were derived from whole blood collected into blood tubes without anticoagulants. These tubes were left at room temperature for 30 minutes prior to centrifugation at 2500 rpm for 10 min at 4°C. Serum aliquots were stored at −80°C for later analysis of IL-22 and HsCRP. Concentration of human IL-22 in serum samples was determined using the ELISA MAXTM Deluxe Set (Biolegend, 434504) using the manufacturer’s instructions. In brief, the plates were coated with the capture antibody for 24 hours at 4°C. Subsequently, plates were washed with PBS-0.05% Tween 20 and non-specific binding was blocked using 1% BSA in PBS (pH 7.4) for 2 hours at room temperature, shaking at 500 rpm. Plates were then coated with serum samples (1:100 dilution) for up to 3 hours, before being detected using the detection antibody, Avidin-HRP and TMB substrate provided. Absorbance was read at 570nm and 450nm within 15 min (data presented as Absorbance 570 nm- 450 nm against the standard curve for hIL-22 provided). Samples were measured in duplicate, with an intra- and inter-assay co-efficient of variation of 1.9% and 13%, respectively. HsCRP was also measured via ELISA kit according to the manufacturer’s instructions (K-ASSAY High-Sensitive C-Reactive Protein kit KAI-160, Kamiya Biomedical).
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