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Quanti blue assay

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QUANTI-Blue assay is a colorimetric assay used to detect and quantify alkaline phosphatase (AP) activity. It provides a simple and sensitive way to measure AP levels in cell culture supernatants, bacterial cultures, or other samples.

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Lab products found in correlation

Raw blue cells, by InvivoGen (7 mentions) Quanti luc assay, by InvivoGen (2 mentions) Synergy h1 plate reader, by Agilent Technologies (2 mentions) Pam3csk4, by InvivoGen (2 mentions) Hek blue il 33 il 1β cells, by InvivoGen (2 mentions) Normocin, by InvivoGen (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) Human xist cdna clone, by OriGene (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Tl8 506, by InvivoGen (1 mentions) 5 ppp dsrna, by InvivoGen (1 mentions) 3 ppp hprna, by InvivoGen (1 mentions) 2 3 cgamp, by InvivoGen (1 mentions) 96 well flat bottom plate, by Corning (1 mentions) Quanti blue solution, by InvivoGen (1 mentions) Synergy 2, by Agilent Technologies (1 mentions) Raw blue, by InvivoGen (1 mentions) Cli 095, by InvivoGen (1 mentions) Human il 6, by R&D Systems (1 mentions)

36 protocols using quanti blue assay

1

Evaluating TLR8-Mediated NF-κB Activation

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The QUANTI-Blue Assay (Invivogen) allows to verify whether a stimulus specific for human TLR8 receptor occurs through the activation of NF-κB. For this purpose HEK-Blue-TLR8 cells (Invivogen) were incubated with Dotap mixtures of synthetic miRNAs or with Lipo246-derived EVs for 24h, then QUANTI-Blue Assay was performed, according to the manufacturer’s instructions.
Casadei L., Calore F., Creighton C.J., Guescini M., Batte K., Iwenofu O.H., Zewdu A., Braggio D.A., Lynn Bill K., Fadda P., Lovat F., Lopez G., Gasparini P., Chen J.L., Kladney R.D., Leone G., Lev D., Croce C.M, & Pollock R.E. (2017). Exosome-derived miR-25-3p and miR-92a-3p stimulate liposarcoma progression. Cancer research, 77(14), 3846-3856.
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2

Quantifying TLR4 Activation in HEK-Blue

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HEK-Blue hTLR4 cells were seeded at 25,000 cells per well in a 96-well plate and treated with liposomes for 2 hours, followed by 16 hours of LPS stimulation. A Quanti-Blue assay (InvivoGen) was performed according to the manufacturer’s instructions to determine the level of secreted alkaline phosphatase as a reporter for TLR4 activation. Cell viability was assessed as mentioned earlier.
Alaarg A., Jordan N.Y., Verhoef J.J., Metselaar J.M., Storm G, & Kok R.J. (2016). Docosahexaenoic acid liposomes for targeting chronic inflammatory diseases and cancer: an in vitro assessment. International Journal of Nanomedicine, 11, 5027-5040.
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3

XIST Regulates NF-κB Activation Assay

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THP-1 and THP1-XBlue cells were transfected with human XIST cDNA clone
(5275 bp, Origene) using Lipofectamine 2000 (Invitrogen) according to the
manufacturer’s protocol. NF-κB activation assay studies were
performed using THP1-XBlue with chromosomal integration of a secreted embryonic
alkaline phosphatase reporter construct that is inducible by NF-κB
(InvivoGen). Briefly, 300 × 103 cells were plated in 6-well
plates and stimulated with LPS 24 hours after transfection with XIST. The
supernatants were collected after 24 h and used for QUANTI-Blue assay
(InvivoGen) to detect alkaline phosphatase as recommended by the manufacturer.
To further confirm the effect of XIST on the nuclear migration
of NF-κB p65 and p52 subunits, ELISA based TransAM®method (Active Motif) was used. Briefly, THP-1 cells were transfected with XIST
plasmids followed by LPS stimulation 24 h later. Nuclei were isolated 4 h after
LPS stimulation as mentioned earlier. Equal amounts of nuclear proteins were
used to determine nuclear p65 and p52 according to the manufacturer’s
protocol.
Shenoda B.B., Ramanathan S., Gupta R., Tian Y., Jean-Toussaint R., Alexander G.M., Addya S., Somarowthu S., Sacan A, & Ajit S.K. (2020). Xist attenuates acute inflammatory response by female cells. Cellular and molecular life sciences : CMLS, 78(1), 299-316.
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4

Dual Reporter Assay for Innate Immunity

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THP1-Dual, THP1-Dual MyD88−/−, and THP1-Dual MAVS−/− cells were seeded in 96-well V-bottom culture plates (Corning) at 75,000 cells per well in antibiotic-free RPMI 1640 growth media with 10% HI-FBS for 3 hours before transfection. A549-Dual and A549-Dual RIG-I−/− cells were seeded in 96-well flat-bottom plates (Corning) at 20,000 cells per well in antibiotic-free DMEM with 10% HI-FBS for 24 hours before transfection. Cells were transfected with mRNA (250 ng per well) or 3′ppp-hpRNA (2500 ng per well; InvivoGen), 5′ppp-dsRNA (2500 ng per well; InvivoGen), or 2′3′-cGAMP (2500 ng per well; InvivoGen) or treated with agonists R848 (2500 ng per well; InvivoGen) or TL8-506 (2500 ng per well; InvivoGen) for 24 hours. Luciferase in the supernatant was measured by the QUANTI-Luc assay (InvivoGen), and SEAP was measured by the QUANTI-Blue assay (InvivoGen) on a Synergy H1 plate reader (BioTek).
Nelson J., Sorensen E.W., Mintri S., Rabideau A.E., Zheng W., Besin G., Khatwani N., Su S.V., Miracco E.J., Issa W.J., Hoge S., Stanton M.G, & Joyal J.L. (2020). Impact of mRNA chemistry and manufacturing process on innate immune activation. Science Advances, 6(26), eaaz6893.
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5

Inflammatory Response Modulation by HDL NP

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J774-dual cells were grown to confluence in T75 flask in selection antibiotics (5 μg/ml Blasticidin and 100 μg/ml Zeocin). 50,000 cells per well were seeded in 96-well plates. The following day, cells were treated with 1 ng/mL LPS (E.coli, O55:B5) and 25 nM HDL NP or miR-HDL NP. After 48 hours, inflammatory response was assessed by measuring SEAP activity by QUANTI-Blue assay according to manufacturer’s protocol (InvivoGen). Briefly, 20 μL of cell supernatant was moved from each well to a new 96 well plate. 180 μL of pre-warmed QUANTI-Blue solution (InvivoGen) was added to each sample. The absorbance was measured at 655 nm in a microplate plate reader (Synergy 2, BioTek).
Wang J., Calvert A.E., Kaplan N., McMahon K.M., Yang W., Lu K.Q., Peng H., Thaxton C.S, & Lavker R.M. (2020). HDL nanoparticles have wound healing and anti-inflammatory properties and can topically deliver miRNAs. Advanced therapeutics, 3(12), 2000138.
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6

Macrophage Activation Assay with Avidin-Silica Beads

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The mouse macrophage cell line RAW-Blue (InvivoGen, Sunnyvale, CA), derived from RAW 264.7, was cultured in media as above, without addition of L929 supplementation. RAW-Blue (1 × 105 cells/ml) were seeded in 96-wells tissue culture plates and inoculated with 3 μm avidin-conjugated silica beads, with or without coating with biotin-PS, for 30 min, and followed by replacing media with or without Pam3CSK4 (50 ng/ml, InvivoGen). Cell activation was determined after 8 h total incubation by the Quanti-Blue assay (InvivoGen), based on optical density measurement of 655 nm.
Dill B.D., Gierlinski M., Härtlova A., Arandilla A.G., Guo M., Clarke R.G, & Trost M. (2015). Quantitative Proteome Analysis of Temporally Resolved Phagosomes Following Uptake Via Key Phagocytic Receptors. Molecular & Cellular Proteomics : MCP, 14(5), 1334-1349.
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7

TLR4 Inhibition in HIV-1-Treated Cells

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TLR4 was inhibited in HIV-1-treated THP1 cells using the TLR4-specific inhibitor CLI-095 (InvivoGen, San Diego, CA), according to the manufacturer's instructions. Briefly, 2.6 μg/ml CLI-095 was added 5 hours prior to PSP treatment. Cells were incubated at 37°C and 5% CO2 during inhibition. After 5 hours, PSP was added at a concentration of 200 μg/ml, following the PSP treatment protocol. TLR4 inhibition was assessed by quantifying cell activation using the QUANTI-Blue assay (InvivoGen, San Diego, CA).
Rodríguez-Valentín M., López S., Rivera M., Ríos-Olivares E., Cubano L, & Boukli N.M. (2018). Naturally Derived Anti-HIV Polysaccharide Peptide (PSP) Triggers a Toll-Like Receptor 4-Dependent Antiviral Immune Response. Journal of Immunology Research, 2018, 8741698.
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8

Evaluating IL-1RA and Antibody Therapy in Cell-Based Assays

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40pg/uL human IL-1RA (PeproTech, US) was pre-incubated with 5ng/uL antibody (G4–21 or isotype control. IL-1RA and antibody were applied to HEK-Blue IL-1β cells (InvivoGen, US) for 2 hours and stimulated with 2pg/uL human IL-1β (PeproTech, US) for 36 hours at 37°C. Alternatively, cells were treated with 8–10 nM IL-1RA and commercial monoclonal (mAbs) or polyclonal antibodies (pAbs) at 40–2000nM, or with patient plasma at 1:10 and 1:20 dilutions, and subsequently stimulated with 0.1–1 nM IL-1β for 24 hours. Cells were treated with antibodies in the presence of IL-1α and IL-1RA, IL-1RA alone, or media, and supernatants assayed using the QUANTI-Blue assay (InvivoGen, hkb-il1b, US).
Human A549 lung epithelial cells and MRC-5 lung fibroblasts (ATCC, US) were treated with 10nM IL-1RA and patient plasma and stimulated with 0.5nM human IL-1α (PeproTech, US) for 24 hours at 37°C. RNA was isolated, cDNA prepared, and samples analyzed using TaqMan FAM-conjugated primer sets. Supernatants were collected and assayed for human IL-6 (R&D, US), IL-8 (R&D, US), and G-CSF (R&D, US) via ELISA.
Jarrell J.A., Baker M.C., Perugino C.A., Liu H., Bloom M.S., Maehara T., Wong H.H., Lanz T., Adamska J.Z., Kongpachith S., Sokolove J., Stone J.H., Pillai S.S, & Robinson W.H. (2021). Neutralizing Anti-Interleukin-1 Receptor-Antagonist Autoantibodies Induce Inflammatory and Fibrotic Mediators in IgG4-Related Disease. The Journal of allergy and clinical immunology, 149(1), 358-368.
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9

Confirming TLR7/8 Targeting of LNP-TR5

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Example 19

In another experiment, in vitro biological activity of LNP-TR5 and to further confirm targeting of TLR7/8, the following experiment(s) were performed using a RAW-Blue™ Cells and QUANTI-Blue™ assay (InvivoGen, San Diego, Calif.). Briefly, Raw Blue™ cells that express human Toll Like Receptors (TLRs) (with the exception of TLR5) and an NF-κB/AP-1-inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene were used. Stimulation of these cells with TR5 lead to NE-κB activation through TLR7/8 which is measured by detection of SEAP levels. RAW-Blue™ Cells were incubated with TR5 and LNP-TR5 at different concentration. After twenty-four (24) hr. incubation with the compounds, TLR stimulation was assessed by measuring the levels of SEAP optimal density (OD) using a QUANTI-Blue™ assay. ODs were normalized to the control (untreated) group. The result showed that treating the cells with TR5 and LNP-TR5 could cause stimulation of TLR7/8 confirming the mechanism of action of TR5 and its activity in LNP form. (See, FIG. 27).

US11679141B2. Formulated and/or co-formulated liposome compositions containing toll-like receptor (“TLR”) agonist prodrugs useful in the treatment of cancer and methods thereof (2023-06-20). Nammi Therapeutics, Inc. [US]. Inventors: David Stover [US], Dhruba Bharali [US], Bruce A Hay [US], Tahmineh Safaie [US].
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10

Confirming TR6 LNP Targeting and Activity

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Example 17

To confirm in vitro biological activity of LNP-TR6 and to further confirm targeting of TLR1/2, the following experiment(s) were performed using a RAW-Blue™ Cells and QUANTI-Blue™ assay (InvivoGen, San Diego, Calif.). Briefly, Raw Blue™ cells that express human Toll Like Receptors (TLRs) (with the exception of TLR5) and an NF-κB/AP-1-inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene were used. Stimulation of these cells with TR6 lead to NF-κB activation through TLR1/2 which is measured by detection of SEAP levels. RAW-Blue™ Cells were incubated with TR6 and LNP-TR6 at different concentration. After twenty-four (24) hr. incubation with the compounds, TLR stimulation was assessed by measuring the levels of SEAP optimal density (OD) using a QUANTI-Blue™ assay. ODs were normalized to the control (untreated) group. The result showed that treating the cells with TR6 and LNP-TR6 could cause stimulation of TLR1/2 confirming the mechanism of action of TR6 and its activity in LNP form, (See, FIG. 25).

US11744874B2. Formulated and/or co-formulated liposome compositions containing toll-like receptor (“TLR”) agonist prodrugs useful in the treatment of cancer and methods thereof (2023-09-05). Nammi Therapeutics, Inc. [US]. Inventors: David Stover [US], Dhruba Bharali [US], Bruce A Hay [US], Tahmineh Safaie [US].
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