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Human il 6 elisa kit

Manufactured by Solarbio
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The Human IL-6 ELISA kit is a quantitative sandwich enzyme immunoassay designed for the in vitro measurement of human interleukin-6 (IL-6) in serum, plasma, and cell culture supernatants. The kit utilizes a specific antibody coated on a 96-well plate to capture IL-6, and a detection antibody linked to an enzyme for colorimetric quantification.

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6 protocols using human il 6 elisa kit

1

Validating Differentially-Expressed Proteins by ELISA

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To confirm these differentially-expressed proteins, ELISA was performed for further verification. In total, 4 × 106 DFCs or iDFCs were grown on 150-mm culture plates and incubated overnight. Cells were washed with PBS and cultured with serum-free DMEM/F12 media. After 24 h treatment, supernatants were collected and centrifuged to remove the debris. Levels of IL-6, TGF-β1 and VEGF were determined using Human IL-6 ELISA kit (Solarbio, China), Human TGF-beta1 Quantikine ELISA Kit and Human VEGF Quantikine ELISA Kit (R&D Systems).
Dou L., Wu Y., Yan Q., Wang J., Zhang Y, & Ji P. (2017). Secretome profiles of immortalized dental follicle cells using iTRAQ-based proteomic analysis. Scientific Reports, 7, 7300.
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2

Quantification of Cytokine Levels

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Concentration of cytokines were determined by using the human IL6 ELISA Kit (SEKH-0013, Solarbio, China) according to the manufacturer’s instructions. In brief, 100 µl of indicated NF medium was incubated with plates at 37℃ for 90 min. Then detection antibody, streptavidin-HRP and TMB were added in order. The absorbance of each well was measured at 450 nm with the SPARK 10 M spectrophotometer (Tecan, Austria).
Zheng S., Hu C., Lin H., Li G., Xia R., Zhang X., Su D., Li Z., Zhou Q, & Chen R. (2022). circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway. Journal of Experimental & Clinical Cancer Research : CR, 41, 71.
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3

Cytokine and Macrophage Marker Detection

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Post treatment, the cultural supernatants of HUVECs in each group were collected and centrifuged at 1000 rpm for 2 min to remove cell debris. Supernatants were harvested for cytokine detection of MCP-1, sICAM-1, and sVCAM-1. The operation steps were strictly carried out according to the instructions of the ELISA kit (human MCP-1 ELISA kit: ab179886, Abcam; human sICAM-1 ELISA kit: 70-EK189-96, MultiSciences; human sVCAM-1 ELISA kit: DVC00, R&D Systems). In addition, ELISA was applied to detect M2 macrophage marker molecules (IL-10) and M1 macrophage marker molecules (IL-6 and TNF-α) in their corresponding supernatants, respectively, as per the instructions of the ELISA kit (human IL-10 ELISA kit: SEKH-0018; Solarbio; human IL-6 ELISA kit: SEKH-001; Solarbio; human TNF-α ELISA kit: SEKH-0047; Solarbio).
Chen F., Ning Y., Liu J., Lian M., Wang J, & Dan H. (2023). miR-147a targets ZEB2 to regulate ox-LDL-induced monocyte adherence to HUVECs, atherosclerotic plaque formation, and stability in atherosclerosis. The Journal of Biological Chemistry, 299(6), 104657.
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4

Quantifying Inflammatory Factors

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At the end of cell incubation with MGO and the treatments, cell supernatants were collected. The levels of the three inflammatory factors in the supernatant were determined using the Human IL-6 ELISA Kit, Human IL-1ß ELISA Kit and Human TNF-α ELISA Kit (Solarbio). The specific steps are detailed in the manufacturer’s instructions.
Chen C., Liu X., Li L., Guo M., He Y., Dong Y., Meng H, & Yi F. (2024). Study of the mechanism by gentiopicroside protects against skin fibroblast glycation damage via the RAGE pathway. Scientific Reports, 14, 4685.
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5

Quantification of Cytokine Levels in Cell Media

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ELISA assays were carried out using R&D Systems kits according to the manufacturer's protocols. Briefly, total protein concentrations in CM were measured using a BCA Protein Assay Kit (Solarbio Science & Technology, Beijing), then CM were diluted to suitable concentrations for measurements of IL-6, IL-8, and TNFα using the following ELISA kits: Human IL-6 ELISA Kit (Cat. No. VAL102, R&D Systems, USA), human IL-8 ELISA Kit (Cat. No. VAL103, R&D Systems, USA) and human TNFα ELISA Kit (Cat. No. VAL105, R&D Systems, USA). Absorbance at 450 nm was measured using a plate reader (Spectrostar Nano, BMG Labtech) and the concentrations of IL-6, IL-8, and TNFα in CM were calculated from the respective standard curves.
Zu T., Wen J., Xu L., Li H., Mi J., Li H., Brakebusch C., Fisher D.E, & Wu X. (2020). Up-Regulation of Activating Transcription Factor 3 in Human Fibroblasts Inhibits Melanoma Cell Growth and Migration Through a Paracrine Pathway. Frontiers in Oncology, 10, 624.
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6

Quantifying Inflammatory Cytokines and Proteases

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The secretion levels of interleukin (IL)-8 and IL-6 were examined by human IL-8 enzyme-linked immunosorbent assay (ELISA) kit (Solarbio, Beijing, China; SEKH-0016) and human IL-6 ELISA kit (Solarbio; SEKH-0013), respectively. All operations were strictly carried out in accordance with the instructions. immunofluorescence Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.3% Triton X-100 and sealed with goat serum for 30 min. Cells were subsequently incubated overnight with antibodies against MMP13 (Proteintech, Wuhan, China; 1:500, 18165-1-AP) and AdAMTS5 (ABclonal, Wuhan, China; 1:200, A2836) for 2 h. Then, cells were incubated with the secondary antibody (Abcam; 1:3000, ab7090) for 1 h, and the nucleus was stained with DAPI (Sangon). Cells were subsequently sealed and observed under a fluorescence microscope (Olympus, Tokyo, Japan).
Huang Y., Pan W., Bao H., Sun X., Xu C, & Ma J. (2024). HSF1 Increases EOGT-Mediated Glycosylation of Notch1 to Promote IL-1β-Induced Inflammatory Injury of Chondrocytes. Cartilage.
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