HEK293 cells expressing human TLR7 or TLR8 with an NF-κB-inducible responsive SEAP reporter gene were obtained from InvivoGen (San Diego, CA, United States). Cells were cultured in DMEM with 10% FBS and antibiotics. Cells were plated at 96-well plates and stimulated for 24 h. Supernatants were harvested and monitored by NF-κB/SEAP activation using HEK-BlueTM Detection Kit (InvivoGen) according to the manufacturer’s instructions.
Hek blue detection kit
Manufactured by InvivoGen
Sourced in United States, France
The HEK-Blue™ detection kit is a tool used to monitor the activation of specific signaling pathways in HEK-Blue™ cells. The kit provides a colorimetric readout to detect the activation of these pathways.
Automatically generated - may contain errors
Lab products found in correlation
Hek blue detection, by InvivoGen (3 mentions)
24 well plate, by BD (2 mentions)
Galacturonic acid, by Merck Group (1 mentions)
Galactose, by Merck Group (1 mentions)
Fucose, by Merck Group (1 mentions)
Rhamnose, by Merck Group (1 mentions)
Mannose, by Merck Group (1 mentions)
Glucuronic acid, by Merck Group (1 mentions)
Xylose, by Merck Group (1 mentions)
Arabinose, by Merck Group (1 mentions)
Glucosamine, by Merck Group (1 mentions)
Cytation 5 cell imaging multi mode reader, by Agilent Technologies (1 mentions)
96 well plate, by BD (1 mentions)
Quanti blue, by InvivoGen (1 mentions)
N acetylcysteine nac, by Merck Group (1 mentions)
Dharmafect 1, by Horizon Discovery (1 mentions)
G 6976, by Merck Group (1 mentions)
Pha 767491, by Selleck Chemicals (1 mentions)
Mk 8776, by Selleck Chemicals (1 mentions)
Azd6738, by MedChemExpress (1 mentions)
12 protocols using hek blue detection kit
1
Quantifying NF-κB Activation in HEK293 Cells
2
Extraction and Characterization of Sparassis latifolia Fungus
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Fruit bodies of Sparassis latifolia were purchased from Fujian Tianyi Industry Co., Ltd. (Ningde city, Fujian Province, China). Monosaccharide standards including fucose, galactose, glucose, arabinose, rhamnose, mannose, glucosamine, xylose, galacturonic acid and glucuronic acid were obtained from SigmaAldrich (St. Louis, MO, USA). Scleroglucan were purchased from Seebio Biotech Co., Ltd. (Shanghai, China). Aniline blue was purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd, (Shanghai, China). HEK-BlueTM hDectin-1b cells and a HEK-BlueTM Detection kit were bought from InvivoGen (Toulouse, France). All other reagents were analytical grade and produced in China.
3
Quantification of SEAP Activity
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HEK-Blue-hTLR4 cells were seeded at a density of 2 × 105 cells/well and grown overnight in 24-well plates (BD Biosciences). The next day, the supernatant was removed, and the medium was replaced with fresh medium. Aliquots of supernatants (200 µl) from the treated cells were transferred to microcentrifuge tubes and heated for 10 min at 65 °C on a heating block (FINEPCR Co., Seoul, Korea). The supernatants were then transferred into new 96-well plates (BD Biosciences), and SEAP production was quantified using the HEK-Blue detection kit (InvivoGen). HEK-Blue IL-1R cells were seeded at a density of 2 × 105 cells/well and grown in 24-well plates (BD Biosciences) overnight, and SEAP production was measured by means of QUANTI-Blue (InvivoGen). Absorbance was measured on a microplate reader (spectrophotometry system; Molecular Devices Inc., Silicon Valley, CA, USA) or a Cytation 5 Cell Imaging Multi-Mode Reader (Bio-Tek Instruments, Winooski, VT, USA) at 620 nm.
4
Colorectal and Breast Cancer Cell Lines
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Colorectal RKO (ATCC), RKOHIF1a+/+ and RKOHIF1a−/− (28 (link)), HCT116, Breast MCF7, MDA-MB231, Bladder T24, VMCUB1 (provided by Prof Anne Kiltie, Oxford University) and HCT116 p53+/+ and p53−/− (provided by Prof Bert Vogelstein, Johns Hopkins Medicine) cells were grown in DMEM media. Non-tumorigenic breast MCF10A (provided by Prof Paul Span, Nijmegen University) cells were grown in MEGM media. Non-transformed lung MRC5 cells (ATCC) were grown in EMEM media. Esophageal OE21 cells (PHE) were grown in RPMI media. All cell media was supplemented with 10% FBS, except MEGM media which was supplemented with 20% FBS, and cells were maintained in an incubator set at 37°C and 5% CO2. All cell lines were verified mycoplasma free using a HEK-Blue™ detection kit (Invivogen). Inhibitors/drugs used were: VX-970 (M6620) (MedChemExpress), AZD6738 (MedChemExpress), Gö6976 (Sigma Aldrich), MK-8776 (Selleckchem), Bay11-7085 (Tocris Biosciences), PHA-767491 (Selleckchem), N’ acetylcysteine (NAC) (Sigma Aldrich), and roscovitine (Ros) (Selleckchem). For siRNA-mediated knockdowns, RKO cells were transfected with siRNA to a final concentration of 50 nM using DharmaFECT 1 (T-2001-03; Horizon Discovery, Cambridge, UK) following manufacturer's protocols. Sequences of siRNAs used: siA3B (CCUGAUGGAUCCAGACACA[dT][dT]), sip53 (GUAAUCUACUGGGACGGAA[dT][dT]), and siATR (CAG GCA CTA ATT GTT CTT CAAd[T]d[T]).
5
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.
6
Quantification of SEAP in HEK-Blue Cells
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HEK-Blue™ hTLR4 cells were seeded at density 2 × 105/well and grown overnight in 24-well plates (BD Biosciences). The cells were treated with different concentrations of TIP3 in the presence and/or absence of LPS. Aliquots of supernatants (200 µl) from the treated cells were transferred to microcentrifuge tubes and heated for 10 min at 65 °C on a heating block (FINEPCR Co., Seoul, Korea). Supernatants were later placed in new 96-well plates (BD Biosciences), and SEAP production was detected with the HEK-Blue™ detection kit (InvivoGen, San Diego, CA, USA). Absorbance was measured on a microplate reader (Molecular Devices Inc., Silicon Valley, CA, USA) at 620 nm.
7
Cell Culture and Transient Transfection
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HeLa, SiHa, HEK293T, U2OS and U2OS pGL13 cell lines were cultivated in DMEM (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum. The cells were grown at 37 °C in a humidified incubator at 5% CO2 (HeLa and SiHa cells) or 10% CO2 (HEK293T, U2OS and U2OS pGL13 cells). Cell cultures were frequently tested and found negative for mycoplasma contamination using the HEK-Blue detection kit from Invivogen. The JetPrime transfection reagent (Polyplus Transfection) was used to transiently express the Nbs in the aforementioned cell lines. Transfection was performed according to the manufacturers’ instructions.
8
NSG Mice Xenograft Melanoma Model
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Female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice were purchased from Charles River laboratory, housed and bred at the UTE animal facility (SFR François Bonamy, IRS‑UN, University of Nantes, license number: B-44-278) under specific pathogen free conditions. Subcutaneous xenograft tumors were established by injection of 1 × 106 M113PD-L1+ or M113WT human melanoma cells in 100 µL PBS (DPBS, ThermoFisher Scientific, Waltham, MA, USA), into the flank of 8–9-week-old NSG mice. Seven days later, when tumor volumes reached around 80 mm3, mice were randomly allocated into the different experimental groups. For each experiment, frozen M113PD-L1+ or M113WT cells were thawed and grown in culture for 10 days before graft. Meanwhile, the absence of mycoplasma contamination was checked using a HEK-Blue Detection Kit (Invivogen, Toulouse, France), and PD-L1 expression was confirmed by flow cytometry analysis.
9
Cytokine-induced TIL activation
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TILs from 22 CRC patients were obtained after culture of tumor fragments for three weeks in RPMI 1640 medium supplemented with 8% human serum, antibiotics, and 150 U/mL IL-2, as previously described [41 (link)]. A weekly check for mycoplasma absence was performed using the HEK-Blue Detection Kit (InvivoGen, San Diego, CA, USA). Then, the obtained TIL cell lines were cultured for 24 h in the presence or absence of recombinant human IL-18 (rhIL-18; B001-5 (MBL, Woburn, MA, USA); 50 ng/mL; 1 × 106 cells in 12-well plates) and IFNγ secreted in culture supernatants was assessed by ELISA as mentioned above.
10
Biological Activity of PEG-Imiquimod Conjugate
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Example 37
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.
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