For IL-1β secretion, THP-1 cells that had been subjected to the differentiation with 50 nM PMA overnight were treated with 2.5 µg/ml LPS for 2 h and then treated with 20 µM nigericin for 30 min. IL-1β measurements were performed using HEK-Blue IL-1β cells (InvivoGen).
Hek blue il 1β cells
Manufactured by InvivoGen
Sourced in United States
HEK-Blue™ IL-1β cells are a cell line derived from human embryonic kidney (HEK293) cells that have been engineered to report on the activation of the IL-1β signaling pathway. These cells stably express an NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene.
Automatically generated - may contain errors
Lab products found in correlation
Quanti blue, by InvivoGen (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Thp 1 cell, by American Type Culture Collection (3 mentions)
Appropriate antibiotics, by Thermo Fisher Scientific (3 mentions)
Transit x2, by Mirus Bio (3 mentions)
Hek293t cell, by American Type Culture Collection (3 mentions)
Hek blue il 1β, by American Type Culture Collection (3 mentions)
Hek blue il 1β reporter cells, by InvivoGen (3 mentions)
Normocin, by InvivoGen (2 mentions)
Zeocin, by InvivoGen (2 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions)
Quanti blue reagent, by InvivoGen (2 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Quanti blue, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Welgene (1 mentions)
Hygromycin, by Thermo Fisher Scientific (1 mentions)
Normocin, by Thermo Fisher Scientific (1 mentions)
Quanti blue detection reagent, by InvivoGen (1 mentions)
23 protocols using hek blue il 1β cells
1
IL-1β Secretion in THP-1 Cells
2
Quantifying IL-1β Receptor Activation
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Production of IL–1β was detected with HEK–Blue™ IL–1β Cells (InvivoGen, France) according to the manufacturer’s instructions. Briefly, Gd–lip, ctrl–lip or Gd–DOTA were added to the suspension of THP1–Null Cells activated by lipopolysaccharide (LPS). After 24 h, THP1 supernatant was added to the suspension of HEK–Blue cells and incubated overnight. Activation of IL–1 receptor was detected by monitoring of the activation of the SAEP reporter gene using QUANTI–Blue™ and quantified by Synergy II spectrophotometer. Recombinant IL–1β (InvivoGen) and ATP were used as positive controls. For more detailed description of the method see60 (link).
3
Quantifying IL-1β Secretion in Macrophages
4
Analyzing IL-1β Neutralizing Activity
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To examine IL-1β neutralizing activity of TA, HEK-Blue IL-1β cells (InvivoGen, San Diego, CA, USA) were used. Cells were cultured in DMEM (WELGENE, Gyeongsangbuk-do, Korea) containing 10% fetal bovine serum (FBS, Corning, Corning, NY, USA), 100 units/ml penicillin, 100 mg/ml streptomycin (Invitrogen), 100 μg/ml Normocin (InvivoGen), and 100 μg/ml Zeocin (InvivoGen), and cultured at 37°C in 5% CO2 humidified incubator. Cells (5×104 cells/well) were seeded in a 96-well plate and stimulated with human recombinant IL-1β (10 ng/ml) with or without various concentrations of TA (0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, or 100 μM). After a 24 h incubation, SEAP activity was detected using QUANTI-Blue™ (Invitrogen) and the OD at 620 nm. Cell viability was measured by analyzing the OD at 450 nm using D-Plus CCK (DONGIN LS, Gyeonggi-do, Korea).
5
Culturing THP-1 and HEK Blue-IL-1β Cells
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THP-1 cells were purchased
from ATCC, and HEK
Blue-IL-1β cells were from Invivogen. THP-1shasc, THP-1shscr,
and THP-1shcasp1 cells were obtained as reported previously.34 (link) THP-1, THP-1shasc, THP-1shscr, and THP-1shcasp1
cells were used at a density of 2 × 106 cells/mL.
These cells were cultured in RPMI 1640 growth media supplemented with
penicillin (50 μg/mL) and streptomycin (50 μg/mL) along
with 10% (v/v) FBS. HEK Blue IL-1β cells were used at a density
of 5 × 105 cells/mL. The cells were cultured in DMEM
media supplemented with glucose (4.5 g/L),l -glutamine (584
mg/L), penicillin (50 μg/mL), streptomycin (50 μg/mL),
Normocin (100 μg/mL), Hygromycin-B Gold (200 μg/mL), and
Zeocin (100 μg/mL). In place of FBS, 10% (v/v) heat-inactivated
FBS was used for cell assays.
from ATCC, and HEK
Blue-IL-1β cells were from Invivogen. THP-1shasc, THP-1shscr,
and THP-1shcasp1 cells were obtained as reported previously.34 (link) THP-1, THP-1shasc, THP-1shscr, and THP-1shcasp1
cells were used at a density of 2 × 106 cells/mL.
These cells were cultured in RPMI 1640 growth media supplemented with
penicillin (50 μg/mL) and streptomycin (50 μg/mL) along
with 10% (v/v) FBS. HEK Blue IL-1β cells were used at a density
of 5 × 105 cells/mL. The cells were cultured in DMEM
media supplemented with glucose (4.5 g/L),
mg/L), penicillin (50 μg/mL), streptomycin (50 μg/mL),
Normocin (100 μg/mL), Hygromycin-B Gold (200 μg/mL), and
Zeocin (100 μg/mL). In place of FBS, 10% (v/v) heat-inactivated
FBS was used for cell assays.
6
Assessing NF-κB Activation via IL-1β Stimulation
7
Evaluating IL-1β Pathway Inhibition
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HEK 293 reporter cells for human IL-1β (HEK-Blue IL-1β cells, Invivogen) were used to test the ability of peptide variants to inhibit IL-1β signaling pathway. HEK-Blue IL-1β cells were used according to manufacturer instructions. HEK-Blue IL-1β cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) additioned with 10% heat inactivated fetal bovine serum (ATCC), 4.5 g/l glucose (Sigma), and 2 mM L-glutamine (ATCC) at 37°C, 5% CO2, in a humidified environment. HEK-Blue IL-1β cells (5,000 cells/well) were plated into 96-well tissue culture plates. The following day, cells were pre-treated for 1 h with peptide variants (Peptides 1.0, 1.1, 1.2, 2.0, 3.0, 4.0, and 4.3) or soluble IL-1RAcP (2 ng/μL) as a positive control. The cells were then stimulated with IL-1β at 2.5 ng/ml for 30 min. Following stimulation, the medium was changed and the inhibitors were added again to the plate. The cells were then left to incubate overnight. The next day, 160 μl of QUANTI-Blue (Invivogen) were added to each well. The plate was incubated at 37°C for 1 h. Then, the plate was placed in a spectrophotometer to detect the levels of secreted embryonic alkaline phosphatase (SEAP) in each well at a wavelength of 625–655 nm. Each peptide was tested in triplicate and an ANOVA test with Dunnett's test for multiple comparisons was performed to determine significance of inhibition.
8
In Vitro IL-1β Neutralization Assay
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MRC-5 cells (ATCC), human lung fibroblasts, were maintained in MEM supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate and 2 mM L-glutamine and used for in vitro neutralization assays as described by Economides et al.13 (link) Briefly, cells were seeded in 96-well plates at 3,000 cells/100 µL/well and incubated overnight at 37 °C in a 5% CO2 humidified incubator. Cells were then treated with 4 pM human IL-1β or 60 pM mouse IL-1β in the presence or absence of various concentrations of test antibodies. Eighteen hours after treatment, IL-1β-induced human IL-6 secretion in the supernatant was measured using the human IL-6 ELISA kit (Biolegend) according to the manufacturer’s protocol. HEK-Blue™ IL-1β cells (InvivoGen) were maintained in DMEM supplemented with 10% FBS, 100 µg/mL Zeocin™ and 200 µg/mL hygromycin B. When used for in vitro neutralization assays, cells were seeded in 96-well plates at 5x104 cells/100 µL/well and incubated overnight at 37 °C in a 5% CO2 humidified incubator. Cells were then treated with 4 pM human IL-1β or 40 pM mouse IL-1β in the presence or absence of various concentrations of test antibodies. Eighteen hours after treatment, IL-1β-induced release of secreted embryonic alkaline phosphatase (SEAP) in the supernatant was assessed using QUANTI-Blue™ (InvivoGen) according to the manufacturer’s protocol.
9
Isolation of Anti-IL-1β Antibodies from Phage Display
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Example 1
Anti-IL-1β antibodies were isolated from a human antibody phage display library (Humanyx HX-02 library) via in vitro selection. IL-1β specific monoclonal antibodies in the Fab format were initially identified by ELISA. These ELISA-positive Fabs were added to HEK-Blue™ IL-1β cells (InvivoGen, USA) which is an engineered HEK293 cell line that overexpresses recombinant human IL-1β receptor.
Binding of IL-1β to its receptor IL-1R on the surface of HEK-Blue™ IL-1β cells triggers a signaling cascade leading to the activation of NF-kB and the subsequent production of secreted alkaline phosphatase (SEAP). Detection of SEAP in the supernatant of HEK-Blue™ IL-1β cells can be readily assessed using QUANTI-Blue™, a colorimetric SEAP substrate. The HEK-Blue cells are sensitive to both human and mouse IL-1β, but are insensitive to human IL-1α or TNF-α.
Of the 22 ELISA-positive Fabs, two clones, 1H and 2H, were found to block the activation of the HEK-Blue IL-1β cells by IL-1β stimulation with IC50 (concentration inhibiting 50% of IL-1β activity) of 1.31 and 0.21 nM, respectively (
10
Isolation of Anti-IL-1β Monoclonal Antibodies
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Example 1
Anti-IL-1β antibodies were isolated from a human antibody phage display library (Humanyx HX-02 library) via in vitro selection. IL-1β specific monoclonal antibodies in the Fab format were initially identified by ELISA. These ELISA-positive Fabs were added to HEK-Blue™ IL-1β cells (InvivoGen, USA) which is an engineered HEK293 cell line that overexpresses recombinant human IL-1 receptor.
Binding of IL-1β to its receptor IL-1R on the surface of HEK-Blue™ IL-1β cells triggers a signaling cascade leading to the activation of NF-kB and the subsequent production of secreted alkaline phosphatase (SEAP). Detection of SEAP in the supernatant of HEK-Blue™ IL-1β cells can be readily assessed using QUANTI-Blue™, a colorimetric SEAP substrate. The HEK-Blue cells are sensitive to both human and mouse IL-1β, but are insensitive to human IL-1α or TNF-α.
Of the 22 ELISA-positive Fabs, two clones, 1H and 2H, were found to block the activation of the HEK-Blue IL-1β cells by IL-1β stimulation with IC50 (concentration inhibiting 50% of IL-1β activity) of 1.31 and 0.21 nM, respectively (
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