Interleukin 6 (il 6)

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IL-6 is a lab equipment product that measures the concentration of interleukin-6 (IL-6), a cytokine involved in various biological processes. The core function of this product is to quantify IL-6 levels in samples.

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Lab products found in correlation

1 655 protocols using interleukin 6 (il 6)

Cytokine Modulation of ARPE-19 Cells

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Cytokines were detected in the supernatants of ARPE-19 cells, seeded at 50,000 cells/well in 24-well plates. ARPE-19 cells were pretreated with 5 ng/ml of different kinds of cytokines (TNF-α (Cat# 300-01A, PeproTech, NJ, USA), IL-6 (Cat# 200-06, PeproTech, NJ, USA), IL-1β (Cat# 200-01B, PeproTech, NJ, USA), TNF-α+IL-6, TNF-α+IL-1β, IL-6+IL-1β, and TNF-α+IL-6+IL-1β) for 10 mins, then stimulated with diacerein (10 and 100 μM/ml), and incubated for 2 hours. Cell-free supernatants were collected at 2 hours after culture and stored at -80°C until further use. Levels of IL-6, IL-8, and MCP-1 were determined using a human IL-6 (Cat# 88-7066-22, Thermo Fisher Scientific, MA, USA), IL-8 (Cat# 88-8086-22, Thermo Fisher Scientific, MA, USA), and MCP-1 (Cat# 88-7399-22, Thermo Fisher Scientific, MA, USA) ELISA Ready-Set-Go kit following the manufacturer's instructions.

Tien P.T., Lin C.H., Chen C.S., Chang C.Y., Ku H., Gan D., Tsai Y.Y., Chen J.J., Lin H.J, & Wan L. (2021). Diacerein Inhibits Myopia Progression through Lowering Inflammation in Retinal Pigment Epithelial Cell. Mediators of Inflammation, 2021, 6660640.

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Cytokine Array Analysis of eMSCs

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Cytokine Array C3 (RayBiotech Inc., Norcross, GA, USA) was used to determine the cytokines in coculture experiments. The signal intensities of the cytokines were quanti ed using the Image J software (NIH Image, National Institutes of Health, USA). A fold change ≥ 3 after coculture was considered as potential cytokine candidate. The chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL5, Granulocytemacrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6), monocyte chemoattractant protein 3 (MCP-3) levels serum free CM collected from coculture experiment of eMSCs with endometrial epithelial and stromal cells from the menstrual phase were determined using enzyme-linked immunosorbent assays (ELISA; IL-6, Invitrogen; CXCL1, CXCL5, GM-CSF and MCP-3, R&D Systems) Four candidate cytokine were shortlisted and recombinant CXCL1 (1000 pg/ml; PeproTech, Rocky Hill, NJ, USA), CXCL5 (600 pg/ml; PeproTech), GM-CSF (500 pg/ml, PeproTech) and IL-6 (500 pg/ml, PeproTech) at concentrations found in the coculture condition was added to the growth medium of the eMSCs seeded at clonal density (500 cell/cm 2 ) for 15 days.

Xu S., Chan R., Li T., Ng E.H.Y., & Yeung W.S. (2020). Understanding the Regulatory Mechanisms of Endometrial Cells on Activities of Endometrial Mesenchymal Stem-Like Cells During Menstruation.

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Cytokine Modulation of eMSC Clonal Growth

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Cytokine Array C3 (RayBiotech Inc., Norcross, GA, USA) was used to determine the cytokines in coculture experiments. The signal intensities of the cytokines were quanti ed using the Image J software (NIH Image, National Insistutes of Health, USA). A fold change ≥ 3 after coculture was considered as potential cytokine candidate. The chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL5, Granulocytemacrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6), monocyte chemoattractant protein 3 (MCP-3) levels serum free CM collected from coculture experiment of eMSCs with endometrial epithelial and stromal cells from the menstrual phase were determined using enzyme-linked immunosorbent assays (ELISA; IL-6, Invitrogen; CXCL1, CXCL5, GM-CSF and MCP-3, R&D Systems) Four candidate cytokine were shortlisted and recombinant CXCL1 (1000 pg/ml; PeproTech, Rocky Hill, NJ, USA), CXCL5 (600 pg/ml; PeproTech), GM-CSF (500 pg/ml, PeproTech) and IL-6 (500 pg/ml, PeproTech) at concentrations found in the coculture condition was added to the growth medium of the eMSCs seeded at clonal density (500 cell/cm 2 ) for 15 days.

Xu S., Chan R., Li T., Ng E.H.Y., & Yeung W.S. (2020). Understanding the Regulatory Mechanisms of Endometrial Cells on Activities of Endometrial Mesenchymal Stem-Like Cells During Menstruation.

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Cytokine and Prostaglandin Quantification by ELISA

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Supernatants obtained from the qPCR assay were centrifuged at 600×g for 6 min and used to estimate concentration using bovine IL-8 ELISA kits (#3114-1A-6, Mabtech, Nacka, Sweden), IL-6 (#ESS0029, Thermo Fisher Scientific), IL-1β (#ESS0027, Thermo Fisher Scientific) and PGE2 (#514010, Cayman Chemicals, Ann Arbor, MI, USA) according to the manufacturer's instructions. Briefly, capture antibody was added to a 96-well plate and incubated overnight. The next day, the wells were blocked for 1 h (4% bovine serum albumin (BSA), 5% sucrose in phosphate-buffered saline (PBS)), and then 100 µL of sample was added and incubated for 1–2 h. After washing the plates twice, the detection antibody was added and incubated for 1 h. After two additional washes, streptavidin was added, and the mixture was incubated for an additional 0.5–1 h. Finally, p-nitrophenyl phosphate (pNPP) or TMB substrate solution was added, followed by incubation for 20–25 min in the dark. All procedures were performed at room temperature. The reaction was stopped with 0.16 M H₂SO₄ (for the IL-6 ELISA kit), and the samples were analyzed at 405, 450, 450 and 420 nm for IL-8, IL-6, IL-1β and PGE2, respectively, in a Varioskan Flash Reader (Thermo Fisher Scientific).

Manosalva C., Alarcon P., Quiroga J., Teuber S., Carretta M.D., Bustamante H., Lopez-Muñoz R., Hidalgo M.A, & Burgos R.A. (2023). Bovine tumor necrosis factor-alpha Increases IL-6, IL-8, and PGE2 in bovine fibroblast-like synoviocytes by metabolic reprogramming. Scientific Reports, 13, 3257.

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Cytokine Response of ARPE-19 and H-RPE Cells

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Cytokines were detected in the supernatants of ARPE-19 and H-RPE cells, seeded at 10,000 cells/well in 96-well plates. ARPE-19 cells were pretreated with 5 ng/ml of different kinds of cytokines IL-6 (Cat# 200–06, PeproTech, NJ, USA), (TNF-α (Cat# 300-01A, PeproTech, NJ, USA), IL-6 + TNF-α for 16 h. Cell-free supernatants were collected and stored at -80 °C until further use. ARPE-19 and H-RPE cells pretreated with 5 ng/ml of cytokines IL-6 + TNF-α for 2 h. Treatment media were subsequently removed and fresh media with or without treatment were applied and incubated for 6 h. Cell-free supernatants were collected and stored at -80 °C until further use. Levels of IL-6, IL-8, and TNF-α were determined using a human IL-6 (Cat# 88–7066-22, Thermo Fisher SCIENTIFIC, MA, USA), IL-8 (Cat# 88–8086-22, Thermo Fisher SCIENTIFIC, MA, USA), and TNF-α (Cat# 88–7399-22, Thermo Fisher SCIENTIFIC, MA, USA) ELISA Ready-Set-Go kit following the manufacturer’s instructions.

Chen C.S., Hsu Y.A., Lin C.H., Wang Y.C., Lin E.S., Chang C.Y., Chen J.J., Wu M.Y., Lin H.J, & Wan L. (2022). Fallopia Japonica and Prunella vulgaris inhibit myopia progression by suppressing AKT and NFκB mediated inflammatory reactions. BMC Complementary Medicine and Therapies, 22, 271.

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Hematopoietic Progenitor Cell Culture

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Isolated CD34 + progenitor cells were cultured, at a final density between 0.1-1 . 10 5 cells/mL, in a serum-free methylcellulose-based medium (MethoCult SF H4236, Stemcell Technologies) supplemented with stem cell factor (SCF, 100 ng/mL) (Miltenyi Biotec, Bergisch Gladbach, Germany) and IL-3 (100 ng/mL) (PeproTech, Rocky Hill, USA), low-density lipoprotein (LDL, 10 μg/mL) (Stemcell Technologies), 2-mercaptoethanol (55 μmol/L) (Thermofischer Scientific, Waltham, USA) and penicillin (100 units/mL) -streptomycin (100 μg/mL) (ThermoFisher Scientific) for two weeks. To study the effect of IL-6, in half of the cultures, IL-6 (50 ng/mL, Peprotech) was added. Thereafter, cells were cultured for an additional two or six weeks in Iscove's Modified Dulbecco's Medium (IMDM, Thermofischer Scientific) containing only SCF (10 ng/mL) or SCF and IL-6 (50 ng/mL) with weekly refreshing of the medium.

Elst J., Sabato V., van der Poorten M.M., Faber M., Van Gasse A.L., De Puysseleyr L.P., Bridts C.H., Mertens C., Van Houdt M., Maurer M., Hagendorens M.M, & Ebo D.G. (2021). Peripheral blood cultured mast cells: Phenotypic and functional outcomes of different culture protocols. Journal of immunological methods, 492.

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Expansion and Isolation of MDSCs

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Mouse MDSCs were expanded from mouse bone marrow harvested by flushing tibias and femurs with media. Following red blood cell lysis, 2.5×10⁶ cells were cultured in 10 mL of RPMI+ 10% FBS augmented with recombinant mouse 40 ng/mL GM-CSF+ 40 ng/mL IL6 (Peprotech) and incubated at 37°C, 5% CO2 3 or 6 days. Vehicle, estradiol, or MPP treatments were added as described above. For 6 day cultures, cytokines and estrogen treatments were refreshed on day 3. Following red blood cell lysis, 2.5×10⁶ cells were cultured in 10 mL of RPMI+ 10% FBS augmented with recombinant mouse 40 ng/mL GM-CSF+ 40 ng/mL IL6 (Peprotech) and incubated at 37°C, 5% CO2 3 or 6 days. Vehicle, E2, or MPP treatments were added as described above. For 6 day cultures, cytokines and E2 were refreshed on day 3. Following incubation, floating and adherent cells were collected, and M-MDSCs and G-MDSCs were isolated via Miltenyi MDSC purification kit according to manufacturer's protocol. Human MDSCs were expanded from human lung cancer patient bone marrow acquired as single cell suspensions (see above). Briefly, 2×10⁶ cells were cultured in 3 mL of IMDM+ 15% FBS supplemented with recombinant human 40 ng/mL GM-CSF+ 40 ng/mL IL6 (Peprotech) and treated with vehicle, 2μM, or 10μM MPP (see above) for 4 days. Cells were subsequently harvested and analyzed by flow cytometry.

Svoronos N., Perales-Puchalt A., Allegrezza M.J., Rutkowski M.R., Payne K.K., Tesone A.J., Nguyen J.M., Curiel T.J., Cadungog M.G., Singhal S., Eruslanov E.B., Zhang P., Tchou J., Zhang R, & Conejo-Garcia J.R. (2016). Tumor Cell-Independent Estrogen Signaling Drives Disease Progression Through Mobilization of Myeloid-Derived Suppressor Cells. Cancer discovery, 7(1), 72-85.

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Estrogen and Progesterone Modulation of IL-6 in ESCs

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ESCs were cultured in DMEM supplemented with 10% FBS. When the cells reached approximately 80% confluence, the medium was removed and replaced with serum-free medium, with addition of 10⁻⁸ mol/l estrogen (E₂), 10⁻⁷ mol/l progestin (P), 10⁻⁸ mol/l E₂ + 10 ng/ml recombinant IL-6 [18 (link)], 10⁻⁷ mol/l P + 10 ng/ml IL-6, 10⁻⁸ mol/l E₂ + 10⁻⁷ mol/l P, or 10⁻⁸ mol/l E₂ + 10⁻⁷ mol/l P + 10 ng/ml IL-6 (PeproTech, Oak Park, CA, USA). The cells were cultured for 24 h, and the supernatant was collected as conditioned medium. For cells treated with IL-6, 2 μg IL-6 neutralizing antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and protein G (Sigma-Aldrich, St. Louis, MO, USA) were sequentially added to the supernatant to neutralize the remaining IL-6 and to precipitate the IL-6/antibody complex, followed by centrifugation at 8000 rpm for 10 min to collect the supernatant.

Nie M.F., Xie Q., Wu Y.H., He H., Zou L.J., She X.L, & Wu X.Q. (2018). Serum and Ectopic Endometrium from Women with Endometriosis Modulate Macrophage M1/M2 Polarization via the Smad2/Smad3 Pathway. Journal of Immunology Research, 2018, 6285813.

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Neonatal Rat Cardiomyocyte Isolation and Stimulation

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Neonatal 1-day-old rat cardiomyocytes were prepared as previously described^{64 (link)}, with minor modifications. Briefly, neonatal rat cardiomyocytes were isolated by enzymatic disassociation of 1-day-old neonatal hearts. Cardiomyocytes were plated separately for 2 h to remove fibroblasts. The cells were resuspended in Dulbecco's modified Eagle's medium containing 4.5 g/l glucose (DMEM, Life Technologies) and supplemented with 5% FBS, 20 μg/ml vitamin B12, 100U/ml of penicillin and 100 μg/ml of streptomycin. After 24 h of plating, the cells were incubated in serum-free medium overnight. For the western blot analysis, the cardiomyocytes were treated with 20 ng/ml IL-6 (Peprotech, Rocky Hill, NJ) or DMSO (Sigma) as a control for 15 min. The cardiomyocytes were pretreated with 100 μM S3I-201 (STAT3 inhibitor) for 2 h and treated with IL-6 and S3I-201 (Merck Millipore) for 15 min. For the immunofluorescence assay, the cardiomyocytes were treated with 20 ng/ml IL-6 (Peprotech, Rocky Hill, NJ) or DMSO as a control for 24 h. The cardiomyocytes were pretreated with 100 μM S3I-201 for 2 h and treated with IL-6 and S3I-201 for 24 h.

Han C., Nie Y., Lian H., Liu R., He F., Huang H, & Hu S. (2015). Acute inflammation stimulates a regenerative response in the neonatal mouse heart. Cell Research, 25(10), 1137-1151.

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Cytokine and Biomarker Quantification

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Blood was collected and allowed to clot for 30 min. Sera were separated and frozen to -20°C within 1 hour of collection and then transferred for storage at -80°C until assayed. Samples were defrosted and IL-6, TNF-α, hsCRP and COMP levels were examined using cytokine-and protein-specific ELISA kits according to the manufacturers' instructions (IL-6: Bender Med-Systems, Austria; TNF-α: Biosource, USA; hsCRP: Biosource, USA; COMP: Anamar, Sweden). All samples of each participant were tested on the same plate to preclude the effect of inter-assay variability.
All samples were tested in duplicates, and the intra-assay and inter-assay coefficients of variation were ≤5%.

Mündermann A., Geurts J., Hügle T., Nickel T., Schmidt-Trucksäss A., Halle M, & Hanssen H. (2017). Marathon performance but not BMI affects post-marathon pro-inflammatory and cartilage biomarkers. Journal of sports sciences, 35(7).

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