Quanti blue

Manufactured by InvivoGen

Sourced in United States, France

QUANTI-Blue is a colorimetric detection reagent used to quantify secreted alkaline phosphatase (SEAP) activity. It is a convenient and sensitive method for monitoring SEAP reporter gene expression in cell culture supernatants.

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Lab products found in correlation

Quanti luc, by InvivoGen (16 mentions) Zeocin, by InvivoGen (13 mentions) Normocin, by InvivoGen (13 mentions) Raw blue cells, by InvivoGen (12 mentions) Pam3csk4, by InvivoGen (9 mentions) Multiskan fc plate reader, by Thermo Fisher Scientific (9 mentions) Blasticidin, by InvivoGen (8 mentions) Opti mem, by Thermo Fisher Scientific (8 mentions) Thp1 xblue cd14 reporter cells, by InvivoGen (7 mentions) Hek blue ifn α β cells, by InvivoGen (6 mentions) Thp1 blue nf κb cells, by InvivoGen (6 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions) Penicillin, by Merck Group (5 mentions) Streptomycin, by Merck Group (5 mentions) Hek blue il 1r cells, by InvivoGen (5 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions) Lipopolysaccharides (lps), by Merck Group (4 mentions) Hek blue il 12 cells, by InvivoGen (4 mentions) Hek blue il 2 cells, by InvivoGen (4 mentions)

Spelling variants of this product

Quanti-Blue detection reagent (9 citations) Quanti-Blue colorimetric assay (4 citations) QUANTI-Blue colorimetric assay reagent (3 citations) QUANTI-Blue media (3 citations) QUANTI-Blue™ reagent solution (2 citations) Quanti-Blue colorimetric enzyme assay kit (2 citations) QUANTI-Blue TM enzyme assay (2 citations) QUANTI-Blue™ buffer (2 citations) QUANTI-Blue™ Detection media (2 citations) QUANTI-Blue alkaline phosphatase detection medium (2 citations)

275 protocols using quanti blue

Assessing Activatable Cytokine Activity

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Example 20

HEK-Blue IL12 cells (InvivoGen) were plated in suspension at a concentration of 250,000 cells/well in culture media with or without 40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL12, chimeric IL12 (mouse p35/human p40) or activatable hIL12 for 24 hours at 37° C. and 5% CO₂. Activity of uncleaved and cleaved activatable hIL12 was tested. Cleaved inducible hIL12 was generated by incubation with active MMP9. IL12 activity was assessed by quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), a colorimetric based assay.

HEK-Blue IL2 cells (InvivoGen) were plated in suspension at a concentration of 50,000 cells/well in culture media with or without 15-40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL2 or activatable hIL2 for 24 hours at 37° C. and 5% CO₂. Activity of uncleaved and cleaved activatable hIL2 was tested. Cleaved inducible hIL2 was generated by incubation with active MMP9 or another protease. IL2 activity was assessed by quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), a colorimetric based assay. Results are shown in FIGS. 59-62.

US11739132B2. Separation moieties and methods of use thereof (2023-08-29). Werewolf Therapeutics, Inc. [US]. Inventors: William Winston [US], Luke Evnin [US], Vinay Bhaskar [US], Giselle Knudsen [US], Daniel J. Hicklin [US], Cynthia Seidel-Dugan [US], Jose Andres Salmeron Garcia [US], Heather R. Brodkin [US].

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Annexin II Peptide Activation of TLR2 and TLR4

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Example 5

TLR2-transfected HEK 293 Blue Cells and TLR4-transfected HEK 293 Blue Cells (Invivogen, San Diego, Calif.) were pulsed with the N-terminal 15 amino acid annexin II fragment peptide (15aa Peptide, SEQ ID NO:5) at different concentrations. Cells were incubated for 48 hours. Following incubation, supernatant was added to Quanti-Blue (Invivogen, San Diego, Calif.) for secreted alkaline phosphatase detection. Results are shown in FIG. 6.

TLR2-transfected HEK 293 Blue Cells and TLR4-transfected HEK 293 Blue Cells (Invivogen, San Diego, Calif.) were pulsed with the N-terminal 15 amino acid annexin II fragment peptide (15aa Peptide, SEQ ID NO:5) or one of the annexin II variants shown in Table 1. Cells were incubated for 48 hours. Following incubation, supernatant was added to Quanti-Blue (Invivogen, San Diego, Calif.) for secreted alkaline phosphatase detection.

US10206986B2. Annexin II variant compositions and methods (2019-02-19). REGENTS OF THE UNIVERSITY OF MINNESOTA [US]. Inventors: John R. Ohlfest [US], Michael R. Olin [US].

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Endotoxicity Screening of Heat-Killed Bacteria

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HEK293 cells stably expressing human Toll-like receptor 4 (hTLR4), myeloid differentiation factor 2 (MD-2), cluster of differentiation antigen 14 (CD14), and a NF-κB-dependent reporter (secreted embryonic alkaline phosphatase) were from InvivoGen (HekBlue human TLR4). Growth conditions and endotoxicity assay were as recommended by the supplier (InvivoGen). Briefly, the desired amounts of heat-killed bacteria were placed in a total volume of 20 μl (diluted in PBS) and distributed in a flat-bottom 96-well plate (BD Falcon). A total of 25,000 HekBlue human TLR4 cells in 180 μl were then added to each well, and the plate was incubated for 20 to 24 h at 37°C and 5% CO₂. Detection of the secreted phosphatase followed the QUANTI-Blue protocol (InvivoGen). The challenged cells (20 μl) were incubated with 180 μl detection reagent (QUANTI-Blue; InvivoGen). The plates were incubated at 37°C and 5% CO₂, and absorbance was measured at 655 nm using a spectrophotometer (Bio-Rad).

Renzi F., Zähringer U., Chandler C.E., Ernst R.K., Cornelis G.R, & Ittig S.J. (2016). Modification of the 1-Phosphate Group during Biosynthesis of Capnocytophaga canimorsus Lipid A. Infection and Immunity, 84(2), 550-561.

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HEK-Blue CD40L SEAP Assay for TNF Activity

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Stimulation of HEK-293 cells by TNF leads to activation of the NF-κB pathway. The HEK-Blue^TM CD40L SEAP (secreted embryonic alkaline phosphatase) reporter cell line used to determine TNF activity (InvivoGen, #hkb-CD40, cells were described as mycoplasma free when purchased and were tested using Mycoalert [Lonza] after each batch of assay vial preparation). Compounds were titrated in DMSO and pre-incubated with TNF at a concentration giving half maximal response (10 pM for human or 20 pM for mouse) or the anti-human TNFR1 agonist antibody (R&D Systems. #AF 225) for 1 h. Cells were added to the compound/stimulus mixture and further incubated for 18 h. SEAP was measured using the colorimetric substrate HEK-Blue Detect^TM or QUANTI-Blue^TM from InvivoGen. Percentage inhibitions for compound dilutions were calculated between a DMSO control and maximum inhibition (by excess anti-TNF biologic, or NF-κB inhibitor–TPCA-1 [Tocris #2559, R&D Systems]). The IC₅₀ was determined as the point of inflexion between the fitted minimum and maximum of the compound dose response curve, calculated using 4PL fitted curve. Mean IC₅₀s are reported as a mean of at least two experiments. Dose response curves for TNF and the anti-human TNFR1 agonist antibody are shown in Supplementary Fig. 9.

O’Connell J., Porter J., Kroeplien B., Norman T., Rapecki S., Davis R., McMillan D., Arakaki T., Burgin A., Fox III D., Ceska T., Lecomte F., Maloney A., Vugler A., Carrington B., Cossins B.P., Bourne T, & Lawson A. (2019). Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer. Nature Communications, 10, 5795.

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Quantifying Immune Responses to E. coli LPS

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Lipopolysaccharides from Escherichia coli 055:B5 (LPS) were ordered from Sigma-Aldrich (St. Louis, MO, United States); Dulbecco’s Modified Eagle Medium (DMEM), 2-Mercapto-ethanol, Penicillin/Streptomycin, and Roswell Park Memorial Institute Medium (RPMI) were ordered from Life Technologies (Auckland, New Zealand); foetal calf serum (FCS) was purchased from Moregate Biotech (Hamilton, New Zealand), DS2 from Tocris Bioscience (Bristol, United Kingdom), Zeocin^TM from Invitrogen (Auckland, New Zealand) and Quantiblue^TM from InvivoGen (CA, United States). The LIVE/DEAD^® Fixable Near-IR Dead Cell Stain was purchased from Thermo Fisher Scientific (MA, United States); Granulocyte-macrophage colony-stimulating factor (GM-CSF) and flow antibodies MHCII FITC, CD80 PE, CD86 PE-Cy7, CD11c BV421, and CD40 APC came from BioLegend (Auckland, New Zealand). The reagents for real-time PCR (qPCR) were purchased from the following suppliers: RNeasy^® Plus Mini kit from Qiagen (Austin, TX, United States), DNA-free^TM kit from Life Technologies Corporation (Carlsbad, CA, United States), SensiFAST cDNA Synthesis Kit and SYBR Green kit from Bioline (London, United Kingdom) and primers ordered from Integrated DNA Technologies (Singapore).

Neumann S., Boothman-Burrell L., Gowing E.K., Jacobsen T.A., Ahring P.K., Young S.L., Sandager-Nielsen K, & Clarkson A.N. (2019). The Delta-Subunit Selective GABAA Receptor Modulator, DS2, Improves Stroke Recovery via an Anti-inflammatory Mechanism. Frontiers in Neuroscience, 13, 1133.

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Quantifying IFNα Neutralization Potency

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The IC50 values of IFNα neutralization of serum samples were tested with the help of HEK-BlueTM IFN-α/β reporter cells (InvivoGen) that express alkaline phosphatase (AP) under the inducible ISG54 promoter after ISGF binding to the IFN-stimulated response elements in the promoter. The cells were grown in DMEM supplemented with heat inactivated 10% FBS and 30 g/ml blasticidin (InvivoGen) and 100 g/ml Zeocin (InvivoGen). Cells were stimulated with IFNα2a (12.5 U/ml, Miltenyi Biotech) that was preincubated for 2 hr with serial dilutions of recombinant antibodies or one fixed concentration (10%) of serum. QUANTI-Blue TM (InvivoGen) colorimetric enzyme assay was used to determine AP in the cell culture supernatants after 21 hr of incubation. OD was measured at 620 nm with Multiscan MCC/340 (Labsystems) ELISA reader and IC50 values were calculated from the dose-response curves using GraphPad Prism eight software.

Hertel C., Fishman D., Lorenc A., Ranki A., Krohn K., Peterson P., Kisand K, & Hayday A. (2019). Response to comment on 'AIRE-deficient patients harbor unique high-affinity disease-ameliorating autoantibodies'. eLife, 8, e45826.

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NF-κB Activation Assay in THP1-XBlue Cells

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THP1-XBlue-CD14 reporter cells (1 × 10⁶ cells/mL) (InvivoGen, San Diego, USA) were seeded in phenol red RPMI (180000 cells/well), supplemented with 10% (v/v) heat-inactivated FBS and 1% (v/v) AAS, and allowed to adhere before they were stimulated with 100 ng/mL E. coli (0111:B4) LPS and with peptides at the indicated concentrations. NF-κB activation was determined after 20 h of incubation according to manufacturer’s instructions (InvivoGen, San Diego, USA). Briefly, activation of NF-κB leads to the secretion of embryonic alkaline phosphatase (SEAP) into the cell supernatant, were it was measured by mixing supernatant with a SEAP detection reagent (Quanti-Blue^TM, InvivoGen), followed by absorbance measurement at 600 nm. Data shown in the peptide concentration range 0–50 μM are mean values obtained from triplicate measurements.

Singh S., Datta A., Schmidtchen A., Bhunia A, & Malmsten M. (2017). Tryptophan end-tagging for promoted lipopolysaccharide interactions and anti-inflammatory effects. Scientific Reports, 7, 212.

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Evaluating NF-κB Activation in Dectin-2 and TLR2 Cell Lines

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The HEK-Blue^TM mDectin-2 and HEK-Blue^TM hTLR2 (InvivoGen), derivatives of HEK293 cells that stably express the murine Dectin-2 or human TLR2 genes respectively, along with a NF-κB-inducible reporter system (secreted alkaline phosphatase) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% Fetal Bovine Serum (FBS, Gibco) 4.5 g/l glucose, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma) and 100 μg/ml zeocin, 200 μg/ml hygromycin, 10 μg/ml blasticidin, 1 μg/ml puromycin and 50 μg/ml mofetil (all from InvivoGen). Mannoconjugates (1 μg/well in isopropanol, except in Figs 4B,D, 5B, 6B and S2C, from 300 ng to 10 ng/well in isopropanol, in Fig. S2D from 20 to 0.2 ng/well) or bacterial cells lysate (1 µg/well in isopropanol) were added to 96-well plates, followed by evaporation of the solvents as previously described. Reporter cells (5 × 10⁴/well) were stimulated for 24 h, after which alkaline phosphatase activity was measured by mixing 20 μl of the culture supernatant and 180 μl of Quanti-Blue^TM (InvivoGen), and reading O.D. at 630 nm.

Decout A., Silva-Gomes S., Drocourt D., Blattes E., Rivière M., Prandi J., Larrouy-Maumus G., Caminade A.M., Hamasur B., Källenius G., Kaur D., Dobos K.M., Lucas M., Sutcliffe I.C., Besra G.S., Appelmelk B.J., Gilleron M., Jackson M., Vercellone A., Tiraby G, & Nigou J. (2018). Deciphering the molecular basis of mycobacteria and lipoglycan recognition by the C-type lectin Dectin-2. Scientific Reports, 8, 16840.

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Modulation of NF-κB Activation by Peptides

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Effects on NF-κB activation were assessed using THP1-XBlue-CD14 reporter cells (InvivoGen, San Diego, CA, USA). Briefly, 180,000 cells well⁻¹ were seeded in 96-well plates in phenol red RPMI media, supplemented with 10% (vv⁻¹) heat-inactivated FBS and 1% (vv⁻¹) Antibiotic-Antimycotic solution (AA). E. coli LPS (100 ng mL⁻¹, Sigma Aldrich, Saint Louis, MO, USA) was then added with and without the peptides GKY25, sGKY25, HVF18, and sHVF18 at different concentrations (1–20 μM). NF-κB activation was determined after 20 h of incubation according to the manufacturer’s instructions (InvivoGen, San Diego, CA, USA) by mixing 20 μL of supernatant with 180 μL of SEAP detection reagent (Quanti-BlueTM, InvivoGen, San Diego, CA, USA), followed by absorbance measurement at 600 nm. Data shown are mean values ± SD obtained from at least four independent experiments, all made in triplicate. In another set of experiments, the cells were stimulated with 100 ng mL⁻¹ of E. coli LPS, 1 μg mL⁻¹S. aureus LTA, 1 μg mL⁻¹E. coli PGN, 1 μg mL⁻¹S. aureus PGN, or 10 μg mL⁻¹S. cerevisiae zymosan in the presence or the absence of 10 μM of linear and stapled HVF18. The experiment was performed as described above.

Petruk G., Puthia M., Samsudin F., Petrlova J., Olm F., Mittendorfer M., Hyllén S., Edström D., Strömdahl A.C., Diehl C., Ekström S., Walse B., Kjellström S., Bond P.J., Lindstedt S, & Schmidtchen A. (2023). Targeting Toll-like receptor-driven systemic inflammation by engineering an innate structural fold into drugs. Nature Communications, 14, 6097.

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IL-23 Signaling Modulation by IL-39

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In order to determine whether IL-39 would interfere with IL-23-induced signaling responses, an IL-23 responsive reporter cell line was purchased (Invivogen). The HEK-Blue^TM IL-23 cells are stably transfected with the human IL-23R gene and a STAT3-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. Cells (5 x 10⁴/well) were stimulated for 20 hrs with various concentrations of recombinant human IL-23 either in the presence or absence of various concentrations of IL-39 IgG1-Fc fusion protein or IgG1-Fc control protein (2–200 pM). The supernatants were then collected and subjected to SEAP determination using QUANTI-Blue^TM according to manufacturer’s instructions (Invivogen).

Ecoeur F., Weiss J., Schleeger S, & Guntermann C. (2020). Lack of evidence for expression and function of IL-39 in human immune cells. PLoS ONE, 15(12), e0242329.

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