Analytical grade reagents of DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS [2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid], chloroauric acid [HAuCl4·3H2O; also known as gold(III) chloride], MTT, LPS from Salmonella enterica, X-tremeGENE siRNA, FuGENE HD transfection reagents, and other reagents were supplied by Sigma-Aldrich (St. Louis, MO, USA) and used as received. A Cytotoxicity Detection kit for determining lactate dehydrogenase content (LDH) was procured from Roche Applied Science (Rotkreuz, Switzerland). The chloromethyl derivative of H2DCFDA (CM-H2DCFDA) was supplied by Thermo Fisher Scientific Inc. (Waltham, MA, USA). Mouse TNF-α, IL-1β, and IL-6 Quantikine ELISA kits were obtained from R&D Systems Inc. (Minneapolis, MN, USA). Primary antibodies against TBP, α-tubulin, iNOS, extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinase (JNK), p38, HO-1, COX-2, Nrf2, IκBα, NF-κBp65, and NQO1 were sourced from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against p-glycogen synthase kinase 3 beta (GSK-3β), GSK-3β, p-p65, p-IκBα/β), IκBα/β), p-IκBα, IκBα, STAT-1, p-STAT-1, p-ERK, p-38, p-JNK, JAK, p-JAK1, p-AMPK, and AMPK were obtained from Cell Signaling Technology (Beverly, MA, USA). siRNAs specific for mouse AMPK, HO-1, Nrf2, and NQO-1 were sourced from Santa Cruz Biotechnology.
Il 6 quantikine elisa kit
Manufactured by R&D Systems
Sourced in United States
The IL-6 Quantikine ELISA kit is a quantitative sandwich enzyme immunoassay designed for the measurement of interleukin-6 (IL-6) levels in cell culture supernates, serum, and plasma. The kit includes a microplate pre-coated with a monoclonal antibody specific for IL-6, a conjugate, and all necessary reagents to perform the assay.
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Lab products found in correlation
Tnf α quantikine elisa kit, by R&D Systems (4 mentions)
Il 1β quantikine elisa kit, by R&D Systems (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Icycler thermal cycler, by Bio-Rad (2 mentions)
Inflammatory cytokine and chemokine multi analyte elisarray, by Qiagen (2 mentions)
Il 6 single analyte elisa kits, by Qiagen (2 mentions)
Vegf a quantikine elisa kit, by R&D Systems (2 mentions)
Vegf quantikine elisa kit, by R&D Systems (2 mentions)
Tnf alpha quantikine elisa kit, by R&D Systems (2 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Santa Cruz Biotechnology (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Chloroauric acid, by Merck Group (1 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (1 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
P jak1, by Cell Signaling Technology (1 mentions)
31 protocols using il 6 quantikine elisa kit
1
DPPH and ABTS Antioxidant Assays for Cytotoxicity and Immune Modulation
2
Quantitative Cytokine Measurement in Serum and Supernatant
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Cytokine measurement in human serum was performed using Human TNF-α and IL-6 Quantikine ELISA kits (R&D Systems, Minneapolis, MN, USA) as per manufacturer’s specifications. Cytokines in cell supernatant were measured using Human TNF-α and IL-6 DuoSet® ELISA kits (R&D Systems, Minneapolis, MN, USA) as per manufacturer’s specifications.
3
Cytokine profiling of antigen-stimulated PBMCs
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PBMCs or splenocytes (>4 × 106 cells) were processed using Lymphoprep on SepMate tubes (StemCell Technologies, MA). Cells were treated with the RBC lysis solution (Sigma) to remove contaminating red blood cells and washed with PBS containing 2% heat-inactivated FBS.
To enhance cytokine expression, PBMCs were stimulated with either AChR (MG-specific antigen, affinity-purified from torpedo electroplax organ) at 1 μg per 1 × 106 cells or a nonspecific stimulant, PMA/ionomycin (Sigma) at 10 ng/ml of medium. Cells were resuspended in RPMI supplemented with 2% heat-inactivated FBS and incubated with AChR, PMA/ionomycin, or PBS for 16 h or 26 h at 37°C in an incubator with 5% CO2 and 95% humidity. The relative levels of induced IL-6 and IL-21, secreted in the culture supernatant, were measured by ELISA (IL-6 Quantikine ELISA kits, R and D Systems, MN; MO IL-21 COATED ELISA, ThermoFisher Scientific, MA) using slight modification of sample dilutions. Cells were lysed to isolate RNA, which was further processed for quantitative RT-PCR to detect the relative abundance of cytokine mRNA. Aliquots of cells were also used for flow cytometry analyses.
To enhance cytokine expression, PBMCs were stimulated with either AChR (MG-specific antigen, affinity-purified from torpedo electroplax organ) at 1 μg per 1 × 106 cells or a nonspecific stimulant, PMA/ionomycin (Sigma) at 10 ng/ml of medium. Cells were resuspended in RPMI supplemented with 2% heat-inactivated FBS and incubated with AChR, PMA/ionomycin, or PBS for 16 h or 26 h at 37°C in an incubator with 5% CO2 and 95% humidity. The relative levels of induced IL-6 and IL-21, secreted in the culture supernatant, were measured by ELISA (IL-6 Quantikine ELISA kits, R and D Systems, MN; MO IL-21 COATED ELISA, ThermoFisher Scientific, MA) using slight modification of sample dilutions. Cells were lysed to isolate RNA, which was further processed for quantitative RT-PCR to detect the relative abundance of cytokine mRNA. Aliquots of cells were also used for flow cytometry analyses.
4
Quantification of Human IL-6R and IL-6 by ELISA
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Human IL-6R and IL-6 cytokine in cell culture supernatants were quantified using IL-6R and IL-6 Quantikine ELISA Kits (R&D Systems Cat# DR600, D6050). One hundred microliters of standards and supernatant (recovered after centrifugation) of HNO-210 cells transduced with miR-449a and inhibitor were added to the wells of a microplate precoated with a monoclonal antibody specific for human IL-6R and IL-6 and incubated for 2 h at room temperature. Each well was filled three times with 400 μL Wash Buffer and 200 μL of an enzyme-linked polyclonal antibody specific for human IL-6R and IL-6 were added to the wells and incubated for 2 h at room temperature. After three washes, 200 μL substrate solution were added to the wells and incubated for 20 min at room temperature. We added 50 μL Stop Solution to each well and determined a color change (from blue to yellow) in proportion to the amount of bound IL-6R and IL-6. The intensity of the wavelength measured on the spectrophotometer was set to 450 nm or 540 nm.
5
Quantification of Serum and Lung Cytokines
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Serum TNF and IL6 levels were quantified with TNF and IL6 Quantikine ELISA Kits (R&D systems, Minneapolis, MN). Cytokine levels were assessed in homogenized lung tissue lysates using IL10 and TGFB1 Quantikine ELISA Kits (R&D systems, Minneapolis, MN). For additional details see online supplementary methods.
6
Quantification of Inflammatory Cytokines
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The protein levels of TNF-α, IL-1β, and IL-6 in the culture media were measured by mouse TNF-α, IL-1β, and IL-6 Quantikine® ELISA kit (R&D Systems, Minneapolis, MN, USA). Briefly, the assay diluent was added into 96-well plate followed by diluted standards, control and samples for 2 h incubation at room temperature. After washed five times with wash buffer, mouse TNF-α, IL-1β or IL-6 conjugate were added and incubated for another 2 h. The substrate solution was then added to the plate after washed with wash buffer. The reaction was stopped using stop solution after 30 min. The optical density was detected using a microplate reader (Tecan Infinite 200, Männedorf, Switzerland) at 450 nm with wavelength correction at 540 nm. The results were calculated based on the standard curve.
7
Quantitative Cytokine Profiling by ELISA
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The level of each protein, TGF-β, IL-1β, IL-10, IL-6 and TNF-α, was determined with ELISA, using the following commercial kits: TGF-β Quantikine ELISA kit (cat. nο. MB100B; R&D Systems, Inc., Minneapolis, MN, USA), IL-1β Quantikine ELISA kit (cat. nο. MLB00C; R&D Systems, Inc.), IL-10 Quantikine ELISA kit (cat. nο. M1000B; R&D Systems, Inc.), IL-6 Quantikine ELISA kit (cat. nο. M6000B; R&D Systems, Inc.) and TNF-α Quantikine ELISA kit (cat. nο. MB100B; R&D Systems, Inc.) Briefly, 100 µl of each standard and sample were incubated with pre-coated antibody plates for 2.5 h at RT with gentle agitation. Following an extensive wash with the provided buffer, 100 µl of biotinylated antibody were added to the wells for 1 h at RT with gentle shaking. The amount of cytokine present in the sample or standard solution present in each well was then determined by incubating the previous mix with 100 µl of streptavidin solution for 45 min at RT and then with 100 µl of TMB One-Step substrate reagent. The colorimetric reaction was stopped after 30 min of incubation at RT by adding 50 µl of stop solution to each well. The absorbance at 450 nm was immediately measured using a microplate reader (Bio-Rad, Hercules, CA, USA).
8
Quantification of IL-6 in Cell Lines
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The SW480 and SW620 cell supernatants were collected to analyze the levels of IL-6. The IL-6 Quantikine ELISA kit (R6000B; R&D Systems, Minneapolis, MN, USA) was used according to the manufacturer’s instructions.
9
Quantification of Gene Expression and Cytokine Levels
10
Quantifying Mouse Inflammatory Markers
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Serum levels of mouse soluble MCP-1 and mouse IL-6 were measured with a Mouse CCL2/JE/MCP-1 or IL-6 Quantikine ELISA Kit (R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer’s protocol.
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