IL-6 levels were monitored using the Human IL-6 ELISA MAX™ Deluxe Set (BioLegend, CA, USA). One day before conducting the ELISA procedure, the capture antibody was diluted in 1 coating buffer A, and then, 100 μL of this solution was added to all 96-well plates. The plate was then sealed and incubated overnight (16–18 h) at between 2 °C and 8 °C. Next, 100 μL of diluted avidin-HRP solution (BioLegend, CA, USA) and 100 μL of TMB substrate solution (BioLegend, CA, USA) were added. After incubating the samples for 15 min, the absorbance was measured at 490 nm using a FlexStation 3 multi-mode microplate reader (Molecular Devices, CA, USA).
Elisa max deluxe set human il 6
Manufactured by BioLegend
Sourced in United States
The ELISA MAX™ Deluxe Set Human IL-6 is a laboratory equipment product that enables the quantitative measurement of human interleukin-6 (IL-6) in biological samples.
Automatically generated - may contain errors
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15 protocols using elisa max deluxe set human il 6
1
Quantifying Human IL-6 Levels
2
Quantification of IL-6 and IL-8 in Cell Culture
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The concentrations of IL-6 and IL-8 in cell culture supernatants were quantified using the Human IL-6 ELISA MAX Deluxe Set (BioLegend, San Diego, CA, USA) and the Human IL-8 ELISA Set (BD Biosciences, Franklin Lakes, NJ, USA) according to manufacturers’ recommendations. Each experiment was performed three times in triplicate.
3
ELISA for Mouse Flt3L and Human IL-6
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ELSA were performed following manufacture protocols. For mouse Flt3L, plasma was tested with mouse Flt3L ELISA Duo Set from R&D systems. For human IL-6, plasma was tested with human IL-6 ELISA MAX Deluxe Set from Biolegend.
4
TNF-α Expression in FLS Inflammation
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To evaluate the TNF-α expression of FLS, a density of 1 × 10 4 cells/ml were seeded in a 24-well plate for ELISA assay. When cells reached the confluence of 80%, the culture medium was suplemmented with 2 µL/mL of interferon-gamma (INF-γ-Thermo Fisher) for 3 days to induce an inflammatory condition in vitro. After that, FLS were treated with 100 µg/mL F.hepatica extract or PBS for 48 h. Concentrations of TNF and IL-6 in supernatants were measured by human TNF-α Elisa Max ™ Standard Set and by human IL-6 Elisa Max ™ Deluxe Set (BioLegend, San Diego, CA, USA). The absorbance of each well was measured at 450 nm and 570 nm in Spec-traMax M3 spectrophotometer. Data analyses were performed in My Assays software ("Four Parameter Logistic Curve" online data analysis tool, MyAssays Ltd, Sussex, UK) and they are representative at pg/mL. Three experiments were performed independently (n = 3).
5
Quantifying IL-6 Induction by SARS-CoV-2 Proteins
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Serially diluted S protein (Spike S1 + S2 ECD-His Recombinant Protein, 40589-V08B1, Sino Biological, Beijing, China), N protein (40588-V08B or 40588-V07E, Sino Biological), or 10 µL of cell lysate suspended in 100 µL of culture medium was added to 100 µL of the K-ML2 cell suspension or monocyte-derived macrophages with 100 µL of culture medium and incubated at 37 °C for 4 h. Then, 500 µL of fresh medium was added to the wells. Two days later, the culture supernatants were harvested, and the levels of IL-6 were measured by an enzyme-linked immunoassay (ELISA MAX Deluxe Set Human IL-6, BioLegend, San Diego, CA).
6
Quantifying Cytokine Levels in PBMC
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The amount of cytokines (CCL2, CXCL8, and Il6) in treated PBMC supernatants were quantified by ELISA using the ELISA MAX Deluxe Set Human CCL2 (Biolegend), ELISA MAX Deluxe Set Human IL8 (Biolegend), and ELISA MAX Deluxe Set Human IL6 (Biolegend) kits according to the manufacturer’s protocol.
7
Cytokine Secretion Profiling of CAR-T Cells
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The E:T = 3:1 in vitro killing conditioned medium (as described in Section 2.4 ) was collected and divided into the T cell group, anti-CD19 CAR-T cell group, single KD anti-CD19 CAR-T cell group, double KD anti-CD19 CAR-T cell group, and tumor cell negative control group (1:1 mixture of K562 and K562-CD19 only). The secretion of IL-6, IFN-γ, IL-17, IL-2 and IL-10 was detected using ELISA MAX™ Deluxe Set Human IL-6 (Biolegend, Cat: 430504), ELISA MAX™ Deluxe Set Human IFN-γ (Biolegend, Cat: 430104), ELISA MAX™ Deluxe Set Human IL-17A kit (Biolegend, Cat: 433914), ELISA MAX™ Deluxe Set Human IL-2 (Biolegend, Cat: 431804), and ELISA MAX™ Deluxe Set Human IL-10 (Biolegend, Cat: 430604), respectively, following the manufacturer’s instructions.
8
Quantifying Cytokine Secretion in Cell Culture
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The concentrations of soluble TNF-α and IL-6 in the cell culture media were determined using ELISA MAX™ Deluxe Set Human TNF-α and ELISA MAX™ Deluxe Set Human IL-6 (both BioLegend, CA, USA), respectively, according to the manufacturer’s protocol.
9
Cytokine Quantification in Cell Cultures
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For cytokine level measurement, the medium suspended CBMCs or PMCs (concentration: 0.5–1 × 106 cells/mL) were incubated with the indicated treatments in a humidified atmosphere with 5% CO2. After incubation, centrifugation (160× g, 5 min, 4 °C) was conducted to collect supernatants, followed by an assessment of IL-6 or TNF by ELISA kits according to their instructions. The used kits were ELISA MAX™ Deluxe Set Human IL-6, ELISA MAX™ Deluxe Set Mouse IL-6, and ELISA MAX™ Deluxe Set Mouse TNF, which were all purchased from Biolegend, San Diego, CA, USA.
10
Cytokine Response to Urban Particle Exposure
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HaCaT cells were seeded at 1 × 105 cells/well in a 24-well plate and incubated for 24 h. Then, cells were exposed to 500 µg/mL of urban particle matter (SRM1648a NISTS) plus the SSE ingredient (2.5 mg/mL) for 24 h. Afterward, medium supernatants were collected and used to evaluate the levels of secreted proinflammatory cytokines IL1-α and IL-6 using an ELISA MAX™ Deluxe Set Human IL-1α and ELISA MAX™ Deluxe Set Human IL-6 (Biolegend, San Diego, CA, USA), respectively. The results are expressed in pg of cytokine/mL. Two independent experiments were performed.
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