HEK-Blue hNOD2 reporter cells (Invivogen, San Diego, CA) were cultured in Dulbecco's modified Eagle's medium (DMEM), 4.5 g/liter glucose, 10% FCS (Omega Scientific, Tarzana, CA) with 50 U/ml penicillin, 50 μg/ml streptomycin, and 100 μg/ml normocin. For antibiotic selection, 30 μg/ml blasticidin and 100 μg/ml zeocin were added. HEK-Blue detection was achieved by adding 2.5 × 104 cells to a 20-μl sample, which was incubated at 37°C in 5% CO2 for 16 h. Secreted embryonic alkaline phosphatase (SEAP) was quantified using HEK-Blue detection (Invivogen, San Diego, CA) and measured by a spectrophotometer at 655 nm.
Hek blue detection
Manufactured by InvivoGen
Sourced in United States
HEK-Blue™ Detection is a colorimetric assay that allows for the detection and quantification of NF-κB, AP-1, and other inducible transcription factor activity. The assay uses a reporter cell line that expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of an inducible promoter containing the relevant transcription factor binding site. When the transcription factor is activated, it binds to the promoter and induces the expression of the SEAP reporter, which can be detected colorimetrically.
Automatically generated - may contain errors
Lab products found in correlation
Hek blue selection, by InvivoGen (5 mentions)
Normocin, by InvivoGen (4 mentions)
Hek blue detection kit, by InvivoGen (3 mentions)
Mouse cd11b antibody, by BioLegend (2 mentions)
Htlr4, by InvivoGen (2 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (2 mentions)
Facsaria 2, by BD (2 mentions)
96 well plate, by Corning (2 mentions)
Zeocin, by InvivoGen (1 mentions)
Blasticidin, by InvivoGen (1 mentions)
Spectramax m2, by Molecular Devices (1 mentions)
Penicillin streptomycin, by Beyotime (1 mentions)
Hek blue htlr4, by InvivoGen (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Hek blue null2 cells, by InvivoGen (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Glucose, by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Fructose, by Merck Group (1 mentions)
20 protocols using hek blue detection
1
HEK-Blue hNOD2 Reporter Cell Assay
2
Evaluation of TLR7/8 Agonists in Reporter Cells
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HEK-BlueTM hTLR7 and hTLR8 cells (InvivoGen, Hongkong, China) were cultured in DMEM supplemented with 10% Fetal Bovine Serum (FBS), Penicillin-Streptomycin (Beyotime, Shanghai, China), blasticidin (InvivoGen), zeocin (InvivoGen), and normocin (InvivoGen). These reporter cells (InvivoGen) were harvested and mixed with HEK-Blue™ Detection (InvivoGen); the mixture was distributed into 96-well plates at 2.5 × 104 cells/well, then treated with dimethyl sulfoxide (DMSO), TLR7/8 (Compound 2) agonist, or Mannose-TLR7/8 (Compound 3 ) at different concentrations (0.006 μM, 0.01 μM, 0.03 μM, 0.06 μM, 0.1 μM, 0.3 μM, 0.6 μM, 1 μM, 3 μM, 6 μM, and 10 μM) for 14 h. We determined secreted embryonic alkaline phosphatase (SEAP) activity by reading the optical density (OD) at 630 nm on a SpectraMax-M2 (Molecular Devices, San Jose, CA, USA) plate reader.
3
TLR4 Activation and Inflammatory Response
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Dialysis tubing D0405-100FT, DPPH, and MTT were purchased from Sigma Aldrich (USA). Monosaccharide reference standards (glucose, galactose, fructose, arabinose, xylose, and mannose), trifluoroacetic acid and TLC silica gel G plates were obtained from Merck (Germany). Dulbecco's modified Eagle's medium (DMEM) was obtained from Gibco Laboratories (Grand Island, NY, USA). The HEK-Blue™ hTLR4, HEK-Blue™ Null2 cells, HEK-Blue™ Detection, and LPS-EB (Standard LPS, E. coli 0111: B4) were obtained from InvivoGen. Human IL-8 ELISA kit was provided by Invitrogen eBioscience (San Diego, CA, USA). The other chemicals and solvent used were in analytical grade.
4
Quantifying TLR9 Activation in HEK-Blue Cells
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HEK-Blue™ hTLR9 cells were seeded at a density of 5 × 10^4 cells per well onto 96-well plates (Corning, Corning, NY, USA) in DMEM supplemented with HEK-Blue™ Detection (Invivogen). The cells in three wells per condition were immediately stimulated with oligonucleotides at 5 µM or water as a solvent control for 18 h. SEAP activity in the media—indicative of NF-κB activity triggered by TLR9 activation—was then determined for each well by measuring the absorbance at 650 nm using a microplate reader, SPECTRA max PLUS (Molecular Devices, Sunnyvale, CA, USA). The obtained TLR9 activity values (n = 3 wells) were normalized to the values of ODN2006 or SY-ODN18-treated cells and presented as mean ± standard deviation (SD).
5
HEK-Blue Cell Stimulation for TLR2/4 Assay
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HEK-Blue cell cultivation and stimulation were carried out as follows. Initially, HEK 293 cells (InvivoGen, San Diego, CA, USA) were cultured in DMEM with 10% fetal bovine serum (vol/vol), penicillin (100 U/mL), streptomycin (100 µg/mL), and Normocin (100 µg/mL) (InvivoGen, San Diego, CA, USA). After the second passage, 1× HEK-Blue selection (InvivoGen, San Diego, CA, USA) was added to the growth medium for TLR2 and TLR4 cells. Cell cultures were refreshed using PBS without centrifugation when the bottles reached 80% confluence. The cells were maintained at 37°C, 5% CO2, and appropriate humidity.
On the day of stimulation, the cells were detached using PBS, counted, and resuspended at a concentration of 140,000 cells/mL for hTLR4 and 280,000 cells/mL for hTLR2 in HEK-Blue Detection (InvivoGen, San Diego, CA, USA). Subsequently, 180 µL of the cell suspension was added to each well of a 96-well plate to be stimulated with 20 µL of purified PGN_1547 protein. The SEAP co-transfection with genes in HEK 293 cells allowed for the colorimetric reaction, which developed over time and was measured at 655 nm absorbance.
On the day of stimulation, the cells were detached using PBS, counted, and resuspended at a concentration of 140,000 cells/mL for hTLR4 and 280,000 cells/mL for hTLR2 in HEK-Blue Detection (InvivoGen, San Diego, CA, USA). Subsequently, 180 µL of the cell suspension was added to each well of a 96-well plate to be stimulated with 20 µL of purified PGN_1547 protein. The SEAP co-transfection with genes in HEK 293 cells allowed for the colorimetric reaction, which developed over time and was measured at 655 nm absorbance.
6
TLR7/8 Activation Assay in HEK Cells
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Add 20 μl various concentrations of TLR agonists (10 dilutions) and in a 96-well plate. Then HEK-Blue hTLR7 and hTLR8 cells were suspended to 4 × 105 cells/ml in HEK-Blue Detection (InvivoGen) medium and added 180 μl per well. The plates were incubated at 37°C in 5% CO2 for 12 ~ 16 h. Stimulation with a hTLR7or hTLR8 ligand activates NF-κB to induce the production of secreted embryonic alkaline phosphatase (SEAP) in the culture supernatant. SEAP could be measured at OD630nm on an ELISA plate reader.
7
Screening for NF-κB Activation Using HEK-Blue Null2-k
8
Evaluating PEG5-NHS-Imiquimod Biological Activity
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Example 22
To test the biological activity of PEG5-NHS conjugated with imiquimod, TLR 7 stimulation cell-based assay was performed using HEK-Blue™ detection kit (InvivoGen, San Diego, USA) per the manufacturer's instruction. HEK-Blue™ hTLR7 cells express two human genes, TLR7 receptor gene and an secreted embryonic alkaline phosphatase (SEAP) reporter gene. Upon interaction with the TLR7 agonist, TLR7 transduces a signal to trigger the activation of NF-κB and to express secreted alkaline phosphatase, which can be detected by using detection medium (HEK-Blue™ detection, a medium used for the quantification of secreted alkaline phosphatase; InvivoGen) and measured with a spectrophotometer.
Briefly, HEK-hTLR7 cells were cultured at a density of 4×104 cells in 96-well plates and maintained in complete DMEM with selective antibiotics, normocin. Cells were stimulated with different concentrations (2-fold dilutions from 20 μg/ml) of imiquimod and the PEG5-NHS conjugated with imiquimod for 18 hours. The activation of TLR7 was analyzed by measuring SEAP from the culture medium using a spectrophotometer at 620 nm.
9
Assessing TLR7 and TLR8 Activation
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HEK-Blue huTLR7 (Invivogen, #hkb-htlr7) and HEK-Blue huTLR8 (Invivogen, #hkb-htlr8) were maintained according to manufacturer’s recommendations. 4×104 HEK-Blue cells were treated with the indicated stimuli in 200 µL HEK-Blue Detection medium/well in a 96-well F bottom plate for 18 hours at 37°C in a CO2 incubator. Secreted embryonic alkaline phosphatase (SEAP) expression was measured using HEK-Blue Detection (Invivogen, #hb-det2) according to manufacturer’s instructions.
10
TLR Agonist Screening in mTLR Cells
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For evaluating TLR stimulation, 20 µl of each treatment was plated in triplicate in 96-well plates. TLR agonist positive controls were run simultaneously: TLR2 (Pam3CSK4, 1 μg/mL), TLR3 (Poly I: C, 1 μg/mL), TLR4 (LPS-EK, 100 ng/mL), TLR5 (FLA-ST, 2.5 μg/mL), TLR7 (CL264, 50 μg/mL), TLR8 (Poly(dT)/Imiquimod 10 μM), TLR9 (CpG ODN 2395, 5 μM), TLR13 (Sa19, 200 μg/mL). 1X PBS was used as a negative control. After plating the treatments, 50,000 mTLR or Null cells suspended in 180 µl HEK-Blue Detection (Invivogen) medium were added to the respective wells. All cells were incubated for 24 hours at 37°C and 5% CO2. Following incubation, TLR stimulation was quantified using a Cytation 3 plate reader (BioTek) measuring absorbance at 620nm.
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