Nucleic acids encoding different formats of IL1RA with aIL6 scFv were cloned into the 3rd generation self-inactivating (SIN) lentiviral transfer vector. 293T cells were transfected with a lentiviral transfer vector as described above by Lipofectamine 2000 (Thermo Scientific). The supernatant from the transfected cells was collected and added to HEK-Blue IL1R Cells (Invivogen) in different dilutions as indicated in the presence of 10 pg/mL IL1B. After overnight incubation, the supernatant of HEK-Blue IL-1R Cells was collected and incubated with substrate solution of Quant-Blue (Invivogen), and the optical absorbance of converted substrate was measured at 650 nm wavelength through a spectrophotometer. The supernatant was also added to HEK-Blue IL6 Cells (Invivogen) in different dilutions as indicated in the presence of 2 ng/mL IL6. After overnight incubation, the supernatant of HEK-Blue IL6 Cells was collected and incubated with substrate solution of Quant-Blue (Invivogen), and the optical absorbance of converted substrate was measured at 650 nm wavelength through a spectrophotometer.
Hek blue il 6 cells
Manufactured by InvivoGen
Sourced in United States
HEK-Blue™ IL-6 cells are a reporter cell line derived from human embryonic kidney (HEK) cells. The cells are engineered to express secreted embryonic alkaline phosphatase (SEAP) under the control of an IL-6-inducible promoter. Stimulation of these cells with IL-6 results in the production and secretion of SEAP, which can be readily detected using a colorimetric or luminescent assay.
Automatically generated - may contain errors
Lab products found in correlation
Quanti blue, by InvivoGen (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Recombinant human il 6, by BioLegend (1 mentions)
Hek blue il 1r cells, by InvivoGen (1 mentions)
Quant blue, by InvivoGen (1 mentions)
Human tnf α quantikine elisa kit, by R&D Systems (1 mentions)
Synergy htx, by Agilent Technologies (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
6 protocols using hek blue il 6 cells
1
Lentiviral IL1RA and IL6 Activation Assay
2
Evaluating Butein's Anti-Inflammatory Effects
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HEK-Blue™ IL-6 cells were obtained from InvivoGen (San Diego, CA, USA). In a 96-well plate, 20 μL butein at various concentrations (0, 3.125, 6.25, 12.5, 25, and 50 μM) were added to the cell suspension (50,000 cells/200 μL). Following this, the recombinant human IL-6 (BioLegend, San Diego, CA, USA) was administered at 10 ng/mL. After 24 h, 20 μL of the IL-6-treated cell suspension was added to 180 μL of QUANTI-Blue™ Solution (JnvivoGen, San Diego, CA, USA) in a new 96-well plate. After 2 h of incubation at 37 °C, the spectrophotometric absorbance between 620 and 655 nm was recorded.
3
Quantification of Cytokine Secretion
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IL-6 and IL-10 in supernatants of THP-1, exposed to GP for 40 h, were detected by the cell-based assays using the human reporter cell line HEK-Blue™ IL-6 cells and HEK-Blue™ IL-10 cells (Invivogen, San Diego, CA, USA), respectively. Both cell lines were maintained in DMEM, supplemented with 10% FBSLE, 2 mM GlutaMAX, Normocin and the selection antibiotics, 250X HEK-Blue™ Selection. HEK-Blue™ cells responded specifically to IL-6/IL-10. The specific detection of bioactive cytokine, via a colorimetric assay of enzyme activity of the expressed reporter gene SEAP, is achieved by binding the specific IL to its receptor on the surface of HEK-Blue™. SEAP was quantified using QUANTI-Blue™ (Invivogen, San Diego, CA, USA), a SEAP detection medium that turns blue in its presence. Absorbance was measured in a microplate spectrophotometer, Synergy HTX (Biotek, Winooski, VT, USA), at 630 nm wavelength. The levels of TNF-α in supernatants of THP-1 exposed to GP for 40 h was detected by using a Human TNF-α Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s protocol.
4
Culturing Esophageal Cancer Cell Lines
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Two human esophageal cancer cell lines, EC109 and CAES17, were purchased from the Cell Resource Center at the Institute of Medical Sciences, Peking Union Medical College. EC109 and CAES17 cells were cultured in RPMI medium 1640 or DMEM, respectively. HEK-Blue™ IL-6 cells were purchased from InvivoGen (San Diego, CA, USA) and cultured in DMEM according to the manufacturer’s instructions. The media for the cell lines were supplemented with 10% FBS (Gibco, Grand Island, NY, USA), 100 µg/mL streptomycin and 100 U/mL penicillin (Hyclone, Logan, UT, USA) and maintained at 37°C in a humidified atmosphere with 5% CO2.
5
PLAG Modulation of IL-6 Signaling
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HEK-Blue IL-6 cells were obtained from InvivoGen (San Diego, CA, USA) and maintained in DMEM supplemented with 10% FBS and 100 U/mL penicillin-streptomycin. Cells (2.5×105/mL) were plated in 48-well plates and treated with IL-6 (5 ng/mL) and/or various concentrations of 0.01, 0.1, 1, or 10 mg/mL PLAG for 24 h. Twenty microliters of culture medium were mixed with 180 μL Quanti-Blue (InvivoGen), and incubated at 37°C for 1 h. Absorbance at 650 nm was measured using an Emax Microplate Reader. Each sample was analyzed in triplicate, and mean values were compared with control and presented as mean ± SD.
6
Inhibition of IL-6 Signaling Pathway
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Reporter cells (HEK-Blue™ IL-6 Cells (InvivoGen, San Diego, CA, USA); 10,000 cells per well in a 96-well plate) were seeded in DMEM with 10% heat-activated FBS (56 °C for 30 min to inactivate the alkaline phosphatase activity) and allowed to fully adhere for 48 h.
After 48 h, the medium was changed. All experiments were performed in three technical replicates. The tested concentrations of Bis-PMSs5 and 6 in the culture medium were 10, 5, 1 and 0.5 µM. After 2 h (time to inhibit the receptor complex), recombinant human IL-6 (10 ng/mL) was added, and cells were further cultured in an incubator at 37 °C and 5% CO2 for 24 h. Tocilizumab (20 ng/mL) in the culture medium was used as a control inhibitor under otherwise identical conditions.
After 24 h of incubation with IL-6, a 20 µL sample was taken from each well following a brief agitation of the culture plate (on a shaker). The sample was mixed with 180 µL of QuantiBlue substrate to quantity the activity of secreted embryonic alkaline phosphatase (SEAP). The optical density (absorbance) of the color product was measured using a microplate reader (SpectrMax iD3 spectrophotometer at 635 nm) after 30 min at 37 °C.
After 48 h, the medium was changed. All experiments were performed in three technical replicates. The tested concentrations of Bis-PMSs
After 24 h of incubation with IL-6, a 20 µL sample was taken from each well following a brief agitation of the culture plate (on a shaker). The sample was mixed with 180 µL of QuantiBlue substrate to quantity the activity of secreted embryonic alkaline phosphatase (SEAP). The optical density (absorbance) of the color product was measured using a microplate reader (SpectrMax iD3 spectrophotometer at 635 nm) after 30 min at 37 °C.
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