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Quanti blue detection reagent

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Quanti-Blue detection reagent is a colorimetric detection system used to quantify alkaline phosphatase (AP) activity. It provides a simple and sensitive method for the colorimetric detection and quantification of AP, which is commonly used as a reporter gene.

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Lab products found in correlation

Triptolide, by InvivoGen (2 mentions) Normocin, by InvivoGen (2 mentions) Flexstation 3, by Molecular Devices (1 mentions) Recombinant flic, by InvivoGen (1 mentions) Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Teader, by Tecan (1 mentions) Scopoletin, by Merck Group (1 mentions) Hek293, by InvivoGen (1 mentions) Recombinant human ifn β standard, by InvivoGen (1 mentions) Hek blue ifn α β cell line, by InvivoGen (1 mentions) Zeocin, by Thermo Fisher Scientific (1 mentions) Hygromycin, by Thermo Fisher Scientific (1 mentions) Normocin, by Thermo Fisher Scientific (1 mentions) Hygrogold, by InvivoGen (1 mentions) Human thp 1 cells, by American Type Culture Collection (1 mentions) Hek blue il 1β cells, by InvivoGen (1 mentions) Thp1 defnlrp3, by InvivoGen (1 mentions) 96 well tissue culture treated plate, by Corning (1 mentions)

Spelling variants of this product

QUANTI-Blue (275 citations) QUANTI-Blue reagent (112 citations)

9 protocols using quanti blue detection reagent

1

Measuring NF-κB Activity in THP1 Cells

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Expression of SEAP can be induced in THP1-Blue cells upon stimulation of cells with LPS [29 ]. SEAP activity, as a measure of NF-κB activity, was determined after 48 h in a colorimetric assay by using Quantiblue detection reagent (Invivogen, Vienna, Austria) at 635 nm according to the manufacturer’s instructions.
Gostner J.M., Raggl E., Becker K., Überalla F., Schennach H., Pease J.E, & Fuchs D. (2015). Bisphenol A suppresses Th1-type immune response in human peripheral blood mononuclear cells in vitro. Immunology letters, 168(2), 285-292.
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2

Flagellin production in transduced T cells

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Primary human CD4+ T cells were transduced with the α-GFP nanobody (LaG17) nanobody synNotch Gal4VP64 receptor and 5× Gal4 response elements controlling S. Typhi FliC expression. 2 × 105 transduced T cells were co-cultured 1:1 with GFP+ or GFP− K562s for 24 hours to induce flagellin production. Cell culture supernatant was harvested 24 hours after stimulation and added to wells containing HEK-Blue hTLR5 reporter cells (Invivogen) that express secreted alkaline phosphatase (SEAP) under control of a TLR5 inducible NF-κB promoter. Recombinant FliC (Invivogen) was utilized as a positive control. Twenty-four hours later supernatant was harvested from HEK-TLR5 cells and alkaline phosphatase activity quantified in a colorimetric assay using QUANTIBLUE detection reagent (Invivogen) with a Flexstation III (Molecular Devices).
Roybal K.T., Williams J.Z., Morsut L., Rupp L.J., Kolinko I., Choe J.H., Walker W.J., McNally K.A, & Lim W.A. (2016). Engineering T cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors. Cell, 167(2), 419-432.e16.
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3

NF-κB Signaling Assay with Triptolide and Scopoletin

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HEK-Blue-Null1 was derived from the human embryonic kidney-293 (HEK-293) cell line and obtained from Invivogen (San Diego, CA, USA). These cells stably express an optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene preceded by the NF-κB promoter.
In a 96-well plate, 5.0 × 104 HEK-Blue-Null1 were incubated overnight at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Dreieich, Germany) supplemented with 2 mM l-glutamine, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 50 units/mL penicillin, 50 µg/mL streptomycin (Gibco) and 100 µg/mL Normocin (Invivogen). Cell were treated with 1.6 nM triptolide (Invivogen) and varying concentrations of scopoletin (5 µM, 20 µM and 40 µM) (Sigma-Aldrich). The activation of NF-κB was induced by 100 ng/mL of TNF-α for 24 h. Then, 20 µL aliquots of cell culture were added to 180 µL of pre-warmed Quanti-Blue detection reagent (Invivogen) per well according to the manufacturer’s instructions. NF-κB activation was detected by measuring SEAP spectrophotometrically at 630 nm (Tecan Teader, Tecan Group Ltd., Maennedorf, Switzerland).
Seo E.J., Saeed M., Law B.Y., Wu A.G., Kadioglu O., Greten H.J, & Efferth T. (2016). Pharmacogenomics of Scopoletin in Tumor Cells. Molecules, 21(4), 496.
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4

IFN-I Bioactivity Assay in Cell Culture

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Human IFN-I bioactivity in tissue culture supernatants was measured with reference to a recombinant human IFN-β standard (InvivoGen) using HEK-BlueIFN-α/β cell line (InvivoGen) and QUANTI-Blue detection reagent, following manufacturer’s instructions. In minireplicon assays, levels of bioactive IFN-I in tissue culture supernatants were determined by evaluating protection capacity against cytopathic effect (CPE) after 24h infection of Vero cells with VSV at moi = 0.1. As control, cells were treated with either 1000, 100 and 10 I.U./ml of IFN-I for 16 hours. Under these conditions, 10 I.U./ml protected against VSV induced CPE.
Loureiro M.E., Zorzetto-Fernandes A.L., Radoshitzky S., Chi X., Dallari S., Marooki N., Lèger P., Foscaldi S., Harjono V., Sharma S., Zid B.M., López N., de la Torre J.C., Bavari S, & Zúñiga E. (2018). DDX3 suppresses type I interferons and favors viral replication during Arenavirus infection. PLoS Pathogens, 14(7), e1007125.
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5

Assessing NF-κB Activation via IL-1β Stimulation

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Human THP-1 cells from the American Type Culture Collection (ATCC, Manassas, VA) were grown at 37°C with 5% CO2 in RPMI-1640 medium supplemented with 10% FBS (v/v), 100 U/ml-100 µg/ml penicillin-streptomycin, and 50 µM β-mercaptoethanol. The NLRP3-deficient THP-1 cells (THP1-defNLRP3, InvivoGen) were grown at 37°C with 5% CO2 following InvivoGen’s instruction in RPMI-1640 medium supplemented with 10% FBS (v/v), 200 µg/ml HygroGold, and 100 µg/ml normocin. HEK-Blue IL-1β cells that contain an IL-1β-sensitive reporter were from InvivoGen and grown following InvivoGen’s instruction at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% FBS (v/v), 4.5 g/l glucose, 2 mM GlutaMAX medium, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml zeocin, 200 µg/ml hygromycin, and 100 µg/ml normocin (all from Life Technologies or InvivoGen).The level of secreted embryonic alkaline phosphatase (SEAP) protein, a truncated form of human placental alkaline phosphatase released into the culture medium, was used as a measurement of NF-κB activation through IL-1β stimulation with the QUANTI-Blue™ detection reagent.
Ruwona T.B., Xu H., Li X., Taylor A., Shi Y.C, & Cui Z. (2016). Towards understanding the mechanism underlying the strong adjuvant activity of aluminum salt nanoparticles. Vaccine, 34(27), 3059-3067.
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6

Amyloid-beta Oligomerization and TLR5 Activation

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1,1,1,3,3,3-hexafluoro-2-propanol (Sigma)–treated Aβ1-40 and Aβ1-42 (Anaspec) were solubilized in DMSO (Sigma). Aggregation was performed in 10 mM HCl, whereas oligomerization was performed in phenol red–free Ham’s F12 media as described earlier (Stine et al., 2003 (link)). TLR5 activation assay was performed with Aβ according to the manufacturer’s instructions. Briefly, HEK-Blue hTLR5 cells (Invivogen), suspended in DMEM and 10% heat-inactivated FBS, were seeded at 40% in 180 µl/well in 96-well tissue culture–treated plates (Costar) 24 h before assay. Flagellin (Invivogen) or Aβ, prepared in endotoxin-free water, was added in duplicate, and cells were incubated at 37°C for 16 h. Alkaline phosphatase secreted to the medium, the measure of TLR5 signaling, was detected using Quanti-Blue detection reagent (Invivogen) by measuring the optical density at 650 nM.
Chakrabarty P., Li A., Ladd T.B., Strickland M.R., Koller E.J., Burgess J.D., Funk C.C., Cruz P.E., Allen M., Yaroshenko M., Wang X., Younkin C., Reddy J., Lohrer B., Mehrke L., Moore B.D., Liu X., Ceballos-Diaz C., Rosario A.M., Medway C., Janus C., Li H.D., Dickson D.W., Giasson B.I., Price N.D., Younkin S.G., Ertekin-Taner N, & Golde T.E. (2018). TLR5 decoy receptor as a novel anti-amyloid therapeutic for Alzheimer’s disease. The Journal of Experimental Medicine, 215(9), 2247-2264.
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7

Measuring NF-κB Activation in Inflammatory Cells

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HEK-Blue-Null1 cells were seeded in a density of (5 × 104/mL) in 96-well plates and incubated overnight at 37° C. Cells were treated with 1 μM triptolide (Invivogen) (positive control) and different concentrations of nimbolide (1 μM, 0.1 μM, 0.01 μM and 0.001 μM) for 1 h. Then, 100 ng/mL TNF-α (Sigma-Aldrich) were added for 24 h to induce the inflammatory state in the cells. To detect NF-κB activation, 20 μL from the supernatant of cell culture were added to 180 μL of pre-warmed Quanti-Blue detection reagent (Invivogen) per well according to the manufacturer’s instructions. NF-κB activation was detected by measuring SEAP spectrophotometrically at 630 nm using a Tecan reader [64 (link)].
Mahmoud N., Saeed M.E., Sugimoto Y., Klauck S.M., Greten H.J, & Efferth T. (2018). Cytotoxicity of nimbolide towards multidrug-resistant tumor cells and hypersensitivity via cellular metabolic modulation. Oncotarget, 9(87), 35762-35779.
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8

Quantifying IL-1 Secretion from Immune Cells

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HEK-Blue IL-1 reporter cells (Invivogen, San Diego, CA), which respond to IL-1 binding to the IL-1R, were incubated in D-10% medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 ug/ml streptomycin, Normocin (100 μg/ml), Hygromycin B Gold (200 μg/ml) and Zeocin (100 μg/ml). For the detection of IL-1 released from THP-1 cells and primary human monocytes, HEK-Blue cells were resuspended at a concentration of 500,000 cells/ml and added to flat-bottom 96-well plates. Supernatants collected from THP-1 cells or primary monocytes were added to the HEK-Blue cells and incubated for 24 hours at 37°C. The HEK-Blue cell supernatant was combined with the QUANTI-Blue detection reagent (Invivogen), incubated at 37°C for 1 to 3 hours, and then quantified with a Spectra Max Plus 384 plate reader (Molecular Devices, San Jose CA) using SoftMax Pro Version 5 (Molecular Devices) software.
Pandori W.J., Lima T.S., Mallya S., Kao T.H., Gov L, & Lodoen M.B. (2019). Toxoplasma gondii activates a Syk-CARD9-NF-κB signaling axis and gasdermin D-independent release of IL-1β during infection of primary human monocytes. PLoS Pathogens, 15(8), e1007923.
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9

Quantifying Recombinant Cytokine Activity

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A 20 μL volume of diluted cytokine control (rIL-12 at 10 ng/mL) or supernatants containing rVSV-mIL12-mGMCSF amplified in two different volumes (T25 and T175 flasks) was added to 50,000 HEK-Blue IL-12 cells seeded in 180 μL of complete HEK-Blue assay media in a 96-well, flat-bottom plate. The plate was incubated for 16 h at 37 °C and 5% CO2. To assess IL-12 activity, the levels of SEAP expression were quantified using a QUANTI-Blue detection reagent (InvivoGen, San Diego, CA, USA), which was prepared as per the manufacturer’s instructions. A 180 μL volume of QUANTI-Blue detection reagent was added to 20 μL of culture supernatant and incubated for 3 h at 37 °C and 5% CO2. Absorbance was measured using a ClarioStar plate reader at 650 nm.
Ryapolova A., Minskaia E., Gasanov N., Moroz V., Krapivin B., Egorov A.D., Laktyushkin V., Zhuravleva S., Nagornych M., Subcheva E., Malogolovkin A., Ivanov R, & Karabelsky A. (2023). Development of Recombinant Oncolytic rVSV-mIL12-mGMCSF for Cancer Immunotherapy. International Journal of Molecular Sciences, 25(1), 211.
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