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Hek blue detection media

Manufactured by InvivoGen
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HEK-Blue detection media is a colorimetric cell culture medium designed for the detection of NF-κB and other inducible transcription factors. It contains a reporter gene that produces a colorimetric signal upon activation of the target transcription factor.

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Lab products found in correlation

Normocin, by InvivoGen (3 mentions) L18 mdp, by InvivoGen (3 mentions) Spectramax plus 384 spectrophotometer, by Molecular Devices (2 mentions) Pam3csk4, by InvivoGen (2 mentions) Zeocin, by InvivoGen (2 mentions) Victor3v plate reader, by PerkinElmer (2 mentions) Hek blue htlr4 cells, by InvivoGen (1 mentions) Alphaimager hp, by Bio-Techne (1 mentions) Blasticidin, by InvivoGen (1 mentions) Fugene, by Promega (1 mentions) Lipopolysaccharides (lps), by InvivoGen (1 mentions) Hek blue mtlr5 cells, by InvivoGen (1 mentions) Bullet blender, by Next Advance (1 mentions) Tlr reporter cell lines, by InvivoGen (1 mentions) Multiskan fc plate reader, by Thermo Fisher Scientific (1 mentions) Lps ek, by InvivoGen (1 mentions) Blasticidin, by Thermo Fisher Scientific (1 mentions) Mg132, by Selleck Chemicals (1 mentions) Spark microplate reader, by Tecan (1 mentions) Hek blue clr selection, by InvivoGen (1 mentions)

18 protocols using hek blue detection media

1

NF-κB Activation in Pneumococcus-Stimulated HEK293T Cells

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HEK 293T cells were seeded in complete DMEM at 104 cells/well in 96 well plates. Cells were transfected with combinations of plasmids as in21 (link). Streptococcus pneumoniae serotype 23F, clinical isolate P1121 was grown and prepared as in13 (link),29 (link). Cells were stimulated 48 h post-transfection using HEK Blue detection media (InvivoGen, San Diego, CA, USA) supplemented with 1 μg/mL of TLR2 agonist Pam3Csk4 (InvivoGen) as a positive control, or heat-killed, lysozyme-digested Streptococcus pneumoniae(multiplicity of infection, MOI = 50). NF-κB activation was measured for Pam3Csk4-stimulated cultures after 24 h and Streptococcus pneumoniae-stimulated cultures after 48 h. Cultures were first analyzed on a Typhoon Trio variable mode imager and quantified using ImageQuant software (ImageMaster, Ann Arbor, MI, USA) to determine GFP expression. The cultures were then read on a SpectraMax 384 Plus spectrophotometer (Molecular Devices) at 655nm absorbance to determine SEAP expression (NF-κB activity). NF-κB activity (Abs630nm) was normalized by dividing SEAP activity by GFP expression.
Novakowski K.E., Huynh A., Han S., Dorrington M.G., Yin C., Tu Z., Pelka P., Whyte P., Guarné A., Sakamoto K, & Bowdish D.M. (2016). A Naturally-Occurring Transcript Variant of MARCO Reveals the SRCR Domain is Critical for Function. Immunology and cell biology, 94(7), 646-655.
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2

Quantifying TLR4-Mediated Inflammatory Response

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HEK-BlueTM hTLR4 cells (InvivoGen, San Diego, CA, USA) were plated at a density of 2.5 × 104 cells/well with Ps-K18 in HEK-Blue detection media (InvivoGen, San Diego, CA, USA). After 1 h, the cells were stimulated by 20 ng/mL LPS and incubated for 8 h at 37 °C in a humidified 5% CO2 atmosphere. SEAP activity was determined by measuring absorbance at 630 nm.
RT-PCR was performed to measure inhibition activity of Ps-K18 on expression of TLR4 mediating inflammatory response signaling as previously reported [55 (link)]. The results visualized by UV illumination using Alpha Innotech gel documentation system (Alphaimager ® HP; Alpha Innotech Corporation, San Leandro, CA, USA).
Jang M., Kim J., Choi Y., Bang J, & Kim Y. (2019). Antiseptic Effect of Ps-K18: Mechanism of Its Antibacterial and Anti-Inflammatory Activities. International Journal of Molecular Sciences, 20(19), 4895.
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3

Evaluating TLR9 Activation by DNA Vaccines

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HEK-Blue cells expressing human or mouse TLR9 (InvivoGen) were cultured in complete DMEM supplemented with 100 µg/ml Normocin, 30 µg/ml Blasticidin and 100 µg/ml Zeocin (all InvivoGen). HEK-Blue cells contain a secreted embryonic alkaline phosphatase (SEAP) reporter system, which generates a colorimetric change following TLR9 engagement.
Transfections were performed with Fugene (Promega), following the manufacturer’s protocol. Cells were plated at 6 × 104 cells per well in a 96-well plate in HEK-Blue detection media (InvivoGen) or complete DMEM. 300 ng of E6E7 DB or PL DNA complexed with Fugene (5 µl/well) was added directly to the cells. In selected wells human A151 (InvivoGen) or mouse 4084-F (InvivoGen) TLR9 antagonists were used at 10 µg/ml. After 42 h A625nm was measured. To confirm transfection efficiency was similar between DB and PL vaccines eGFP encoding constructs were used, with eGFP expression assessed using a fluorescent microscope.
To investigate the involvement of cytosolic DNA sensing pathways, the THP1-Blue ISG reporter cell line and the THP1-Blue ISG-KD-STING cell line which has STimulator of INterferon Genes (STING) knockdown, were used (both InvivoGen). Transfections were performed as above and 24 h later the cell supernatants  were incubated with QUANTI-Blue reagent for 25 min and A625nm was measured.
Allen A., Wang C., Caproni L.J., Sugiyarto G., Harden E., Douglas L.R., Duriez P.J., Karbowniczek K., Extance J., Rothwell P.J., Orefo I., Tite J.P., Stevenson F.K., Ottensmeier C.H, & Savelyeva N. (2018). Linear doggybone DNA vaccine induces similar immunological responses to conventional plasmid DNA independently of immune recognition by TLR9 in a pre-clinical model. Cancer Immunology, Immunotherapy, 67(4), 627-638.
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4

Quantifying TLR9 Signaling in HEK-Blue Cells

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HEK-Blue mTLR9 reporter cells were plated at 5 × 105 cells/well in HEK-Blue Detection Media per the manufacturer’s instructions (InvivoGen). Control samples included media only, a vehicle control (PBS), and cells treated with a TLR4 agonist, lipopolysaccharide (LPS, 0.2 μg/well, InvivoGen) to verify TLR9-specific reporter signal. All remaining wells were treated with 1 μg/well of soluble CpG. (Poly1/GpG)100 and (Poly1/CTRL)100 PEMs were incubated in 1 mL of 1 X PBS at 37°C with agitation for the indicated amount of time, as described above, and release solutions were added to CpG-treated wells. Matched doses of soluble GpG and soluble CTRL were also prepared and added to CpG-treated wells. Cultures were incubated for 18 hours and TLR9 signalling was quantified measuring the absorbance at 620 nm. Relative TLR9 signalling was determined by normalizing absorbance values to the average absorbance of control wells treated with media only.
Tostanoski L.H., Eppler H.B., Xia B., Zeng X, & Jewell C.M. (2019). Engineering release kinetics with polyelectrolyte multilayers to modulate TLR signalling and promote immune tolerance. Biomaterials science, 7(3), 798-808.
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5

Quantifying NOD2-mediated NF-κB Activation

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HEK-Blue cells expressing human NOD2 and NFkB-SEAP reporter (Invivogen) were seeded into 96 well clear plates at 7.5x103 cells per well in 100 μL of DMEM media supplemented with 10% FBS and 1% antibiotic-antimycotic mix. Cells were allowed to attach for 48 h in 5% CO2 tissue culture incubator at 37 °C. On the morning of the experiment, media in the wells was replaced with 100 μL of HEK-Blue detection media (Invivogen). Cells were treated with the inhibitors, diluted in DMSO (0.5 μL per well) for 15 min in 5% CO2 tissue culture incubator at 37 °C. After that, cells were stimulated by the addition of 1 ng/well L18-MDP (Invivogen). Cells were incubated in 5% CO2 tissue culture incubator at 37 °C for 8 h and absorbance, corresponding to the SEAP in the media, was determined in Wallac3V plate reader (Perkin Elmer). Inhibition, %= (1-((sample signal-unstimulated and DMSO treated cells)/(L18-MDP stimulated and DMSO treated cells - unstimulated and DMSO treated cells)))*100. IC50 values were calculated based on a dose range of inhibitor concentrations using non-linear regression in GraphPad Prism software.
Nikhar S., Siokas I., Schlicher L., Lee S., Gyrd-Hansen M., Degterev A, & Cuny G.D. (2021). Design of pyrido[2,3-d]pyrimidin-7-one inhibitors of receptor interacting protein kinase-2 (RIPK2) and nucleotide-binding oligomerization domain (NOD) cell signaling. European journal of medicinal chemistry, 215, 113252.
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6

Fecal Flagellin-Induced Inflammatory Assay

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Fecal samples were resuspended in phosphate-buffered saline (PBS) to a concentration of 100 mg and then homogenized for 5 min using a bullet blender (Next Advance, Averill Park, NY, USA) without the addition of beads, followed by 2 min centrifugation at 8000× g. Reporter cell suspension was made with HEK-BLUE-mTLR5 cells and HEK-BLUE detection media (Invivogen, San Diego, CA, USA) and applied to fecal homogenates according to manufacturer’s instructions. Purified flagellin (Enzo Life Sciences, Farmingdale, NY, USA) isolated from Salmonella typhimurium was used as an experimental standard. After 24 h of incubation, alkaline phosphatase activity was measured via spectrophotometry absorbance at 635 nm. Serum MPO levels were assayed using Duoset mouse myeloperoxidase ELISA kit (R&D Systems, Minneapolis, MN, USA).
Wu B., Bhatnagar R., Indukuri V.V., Chopra S., March K., Cordero N., Chopra S, & Reddivari L. (2020). Intestinal Mucosal Barrier Function Restoration in Mice by Maize Diet Containing Enriched Flavan-4-Ols. Nutrients, 12(4), 896.
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7

TLR Reporter Cell Assay

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The TLR reporter cell lines (Invivogen) were cultured in DMEM containing 10% FBS and 1× antibiotics. When the HEK-Blue™ TLR4 reporter cells reached 80% confluency, they were harvested using a scraper and resuspended in HEK-Blue™ Detection media (InvivoGen). The resuspended cells (5 × 104 cells/well; 180 μl) were then incubated with 20 μl of each stimulant in a 96-well plate. After 20 h of culture, the optical density at 630 nm (OD630) was measured.
Kim C.U., Lim D., Kim Y.S., Ku B, & Kim D.J. (2023). Influenza viral matrix 1 protein aggravates viral pathogenicity by inducing TLR4-mediated reactive oxygen species production and apoptotic cell death. Cell Death & Disease, 14(3), 228.
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8

Assessing Endotoxin Contamination in CNCs

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HEK mTLR4 cells were used as a semiquantitative readout of endotoxin contamination.52 (link) CNCs were first washed three times with endotoxin-free water. For each wash, CNCs samples were dispersed at approximately 1000 μg/mL in water, ultrasonicated for 5 min (3 s on/off cycles at 25% power), and subjected to ultracentrifugation at 10 000 g to precipitate the CNCs. The solutions were decanted and redispersed after each wash and freeze-dried at the end of cleaning. CNC concentrations of 10–100 μg/mL were then used to determine the contamination of the CNC samples. Particle suspensions of 100 and 1000 μg/mL were prepared in endotoxin-free water, and 20 μL of each suspension was plated in triplicate in a 96-well plate. LPS-EK (InvivoGen) with a known endotoxin content was used as a positive control. HEK Blue detection media (InvivoGen) was reconstituted in 50 mL of endotoxin-free water, heated to 37 °C for 30 min, and passed through a 0.22 μm pore-diameter filter. HEK Blue mTLR4 cells were passaged and plated in the 96-well plate at 25 000 cells/well in 180 μL of HEK Blue detection media. Cells were incubated with the CNCs at 37 °C and 5% CO2 overnight. The plate was analyzed after 12 h using a Multiskan FC plate reader (Thermo Scientific), and the absorbance was measured at 620 nm.
Weiss A.M., Macke N., Zhang Y., Calvino C., Esser-Kahn A.P, & Rowan S.J. (2021). In Vitro and in Vivo Analyses of the Effects of Source, Length, and Charge on the Cytotoxicity and Immunocompatibility of Cellulose Nanocrystals. ACS biomaterials science & engineering, 7(4), 1450-1461.
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9

Quantifying TLR3 Activation in HEK-Blue Cells

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HEK-Blue hTLR3 cells (Invivogen, Cat. No. hkb-htlr3), cells in which native Tlr3 is expressed in endosomes, were grown to a confluency of at least 85%, as described by the manufacturer. Subsequently, cells were harvested and diluted to a concentration of 5 × 105 cells/ml in HEK-Blue growth medium (DMEM supplemented with 10% FBS, penicillin-streptomycin, 100 μg/ml of noromycin, 30 μg/ml of blasticidin (Fisher Scientific, Cat. No. MT30100RB), 100 μg/ml of zeocin (Invivogen, Cat. No. ant-zn-1), 10 μM HEPES, and 10 μM MG-132 (Selleck Chemicals, Cat. No. S2619)). Cells were seeded in a flat-bottom 96-well plate at a final concentration of 5 × 104 cells/well, and 20 μl of stimulus was added (PBS or 109 phage/ml). Cells were incubated for 16 hours at 37°C with 5% CO2, after which the media was replaced with 100 μl of HEK-Blue growth media. After 8 hours incubation, 100 ml of HEK-Blue Detection media (Invivogen, Cat. No. hb-det2) was added. After a final 16-hour incubation, discoloration of the media was measured at 620 nm using a Spark microplate reader (Tecan).
Sweere J.M., Van Belleghem J.D., Ishak H., Bach M.S., Popescu M., Sunkari V., Kaber G., Manasherob R., Suh G.A., Cao X., de Vries C.R., Lam D.N., Marshall P.L., Birakova M., Katznelson E., Lazzareschi D.V., Balaji S., Keswani S.G., Hawn T.R., Secor P.R, & Bollyky P.L. (2019). Bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection. Science (New York, N.Y.), 363(6434), eaat9691.
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10

Dectin-1a Signaling Pathway Assay

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HEK-Blue hDectin-1a cells (InvivoGen; catalog hkb-hdect1a) were maintained in growth medium containing DMEM (4.5 g/L glucose), 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μg/mL Normocin (InvivoGen; catalog ant-nr-05), 2 mM l-glutamine, 1 μg/mL puromycin (InvivoGen; catalog ant-pr-1), and 1× HEK-Blue CLR Selection (InvivoGen; catalog hb-csm). This cell line expresses the Dectin-1a isoform and genes involved in the Dectin-1/NF-κB/SEAP signaling pathway. On the assay day, 180 μL/well of cells at a concentration of 2.8 × 105 cells/mL in HEK-Blue Detection media (InvivoGen; catalog hb-det2) were plated in a 96-well tissue culture plate. Plasma samples (20 μL) were added to each well with and without piceatannol (250 nM). The plates were then incubated at 37°C and 5% CO2 for 24 hours. Levels of SEAP were monitored and measured spectrophotometrically at 620 nm. As controls, 20 μL of water, piceatannol (250 nM), 2 μL of DMSO (piceatannol solvent), 20 μL of 10 μg/mL β-glucan peptides (InvivoGen; catalog tlrl-bgp), or 20 μL of β-glucan peptides + piceatannol (250 nM) were added in separated wells.
Giron L.B., Peluso M.J., Ding J., Kenny G., Zilberstein N.F., Koshy J., Hong K.Y., Rasmussen H., Miller G.E., Bishehsari F., Balk R.A., Moy J.N., Hoh R., Lu S., Goldman A.R., Tang H.Y., Yee B.C., Chenna A., Winslow J.W., Petropoulos C.J., Kelly J.D., Wasse H., Martin J.N., Liu Q., Keshavarzian A., Landay A., Deeks S.G., Henrich T.J, & Abdel-Mohsen M. (2022). Markers of fungal translocation are elevated during post-acute sequelae of SARS-CoV-2 and induce NF-κB signaling. JCI Insight, 7(15), e160989.
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