For detecting IFN, the supernatants from type I IFN–producing cells were added to B16-Blue IFN-α/β cells in a 96-well plate. After a 24-h incubation, the supernatants were assayed for secreted embryonic alkaline phosphatase (SEAP) activity using QUANTI-Blue reagent (InvivoGen) according to the manufacturer’s instructions. Briefly, 50 μl of cell supernatant containing SEAP was mixed with 150 μl of QUANTI-Blue reagent and incubated for 3–4 h at 37°C, and absorbance was measured at 650 nm using a spectrometer. The concentrations of type I IFN in test samples were quantified using a standard curve generated from recombinant type I IFN-β–treated B16-Blue IFN-μ/β cells in the same assay. For assessing TLR7 or TLR9 activation by CLs, HEK-Blue null, HEK-Blue human TLR7, and HEK-Blue human TLR9 reporter cells (InvivoGen) were stimulated with indicated concentrations of R-DOTAP, PMA (Sigma), R848 (InvivoGen), ODN2006 (InvivoGen), or ODN2395 (InvivoGen) for 24 h. After stimulation, cell supernatants were assayed for TLR and NF-κB–induced SEAP activity using QUANTI-Blue reagent according to the manufacturer’s instruction.
Quanti blue reagent
Manufactured by InvivoGen
Sourced in United States, France, Switzerland
QUANTI-Blue reagent is a colorimetric detection system used to quantify alkaline phosphatase (AP) activity. It provides a simple and accurate method for the measurement of AP levels in cell culture supernatants.
Automatically generated - may contain errors
Lab products found in correlation
High glucose dmem, by Thermo Fisher Scientific (16 mentions)
Fetal bovine serum (fbs), by Merck Group (16 mentions)
Hek blue tlr cells, by InvivoGen (15 mentions)
Raw blue cells, by InvivoGen (10 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
M200 microplate reader, by Tecan (3 mentions)
Enspire plate reader, by PerkinElmer (3 mentions)
Hek blue ifn α β indicator cells, by InvivoGen (2 mentions)
Lps from salmonella minnesota s form, by Enzo Life Sciences (2 mentions)
Microplate reader, by Bio-Rad (2 mentions)
Celltiter 96 aqueous one solution, by Promega (2 mentions)
Normicin, by InvivoGen (2 mentions)
Lumikine xpress mifn β elisa kit, by InvivoGen (2 mentions)
Lsrfortessa, by BD (2 mentions)
Easysep mouse nk cell isolation kit, by STEMCELL (2 mentions)
Hek blue null1 cells, by InvivoGen (2 mentions)
Hek blue il 1β cells, by InvivoGen (2 mentions)
Seap reporter gene, by InvivoGen (2 mentions)
Infinite 200, by Tecan (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
112 protocols using quanti blue reagent
1
Quantifying Type I IFN and TLR Activation
2
Quantifying Interferon Levels in Virus-Inactivated Supernatant
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Media were removed from cells before treatment with Universal IFN-I or human IL-29/IFN Lambda 1 (IFN-III) (both from PBL Assay Science, Piscataway, NJ, USA) diluted in DMEM supplemented with 2% HI FBS and at the concentrations indicated in the figure legends. Prior to quantifying biological levels of IFN-I from the supernatant of infected cells, inactivation of virus was performed. Briefly, the indicated supernatant was acid treated with 1N HCl at room temperature for 30 min, followed by titration with 1N NaOH to achieve neutralization of acid. IFN-I was subsequently quantified in virus-inactivated supernatant using HEK-Blue™ IFN-α/β indicator cells (Invivogen) and QUANTI-Blue™ reagent (Invivogen), as previously described [11 (link)].
3
Evaluating NF-κB Activation in Macrophages
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Raw-Blue cells (Invivogen), which are a derivative of RAW 264.7 cells, a mouse macrophage-like line containing a stably incorporated NFκB reporter construct, were used to assess NFκB activation indirectly by measuring secreted alkaline phosphatase activity (SEAP) using the QuantiBlue reagent, as per the manufacturer’s (InvivoGen) instructions. We assessed activation by multiple concentrations of PgLPS (data not shown) and found that 0.1μg/ml was sufficient for robust, consistent activation (abs 635 = ~0.5 within 45 minutes), without rapid color saturation, and used this concentration throughout. Cells were pre-treated for 15 minutes with anti-TLR2 antibody (0.5μg/ml final) or anti-CD36 antibody (12.5μg/ml final). LDL and oxLDL were added 30 minutes prior at a concentration of 25μg/ml. These concentrations of anti-CD36 antibody, LDL and oxLDL were also used in experiments demonstrating decreased pyroptosis. Cells were pre-treated with thrombospondin-1 (TSP) for 15 minutes at a concentration of 50–500nm (a range that has been shown to inhibit angiogenesis). Although not shown, we also pre-treated with TSP for 30 minutes, 2 hours, or treated cells with TSP and PgLPS simultaneously. No differences were observed in SEAP activity.
4
Quantifying SEAP Levels by Colorimetry
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30 μl of the supernatant was mixed with 200 μl of QUANTI-Blue reagent (InvivoGen), and the mixture was incubated at 37 °C for 24 h. The levels of SEAP were measured at an absorbance at 625 nm (A625).
5
Evaluating TLR4 Activation by Heat-Inactivated Bacteria
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In vitro stimulation of TLR4 activity by heat-inactivated bacteria was assessed in a similar method as previously described66 (link). Briefly, bacteria were harvested, washed once in PBS−/− and resuspended in PBS−/− to an OD600 of 5, before being heat-inactivated at 80 °C for 1 h. Bacterial growth was tested to confirm efficient heat inactivation. HEK-Blue hTLR4 and Null2 cells (InvivoGen) were seeded at 50,000 cells in 100 μl per well of a 96-well plate and grown in complete growth medium as described above. Twenty-four hours after seeding, 100 μl of complete growth medium were added. Forty-eight hours after seeding, cells were washed once in complete growth medium without selection antibiotics and stimulated with 100 μl heat-inactivated bacteria in complete growth medium without selection antibiotics at an OD600 of 0.5. Twenty-four hours post stimulation, the supernatant was cleared by centrifugation at 740g for 10 min at room temperature and assayed for NFκB-responsive, secreted alkaline phosphatase activity using QUANTI-Blue reagent (InvivoGen). A650 was assessed at 37 °C. The averages of the cell-free medium and the Null2 cell controls were subtracted to obtain TLR4 activation of HEK-Blue hTLR4 cells.
6
TLR7 Agonist Activity Assay
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Example 4
This example describes a method for assaying TLR7 agonist activity of the compounds disclosed in this specification.
Engineered human embryonic kidney blue cells (HEK-Blue™ TLR cells; Invivogen) possessing a human TLR7-secreted embryonic alkaline phosphatase (SEAP) reporter transgene were suspended in a non-selective, culture medium (DMEM high-glucose (Invitrogen), supplemented with 10% fetal bovine serum (Sigma)). HEK-Blue™ TLR7 cells were added to each well of a 384-well tissue-culture plate (15,000 cells per well) and incubated 16-18 h at 37° C., 5% CO2. Compounds (100 nl) were dispensed into wells containing the HEK-Blue™ TLR cells and the treated cells were incubated at 37° C., 5% CO2. After 18 h treatment ten microliters of freshly-prepared Quanti-Blue™ reagent (Invivogen) was added to each well, incubated for 30 min (37° C., 5% CO2) and SEAP levels measured using an Envision plate reader (OD=620 nm). The half maximal effective concentration values (EC50; compound concentration which induced a response halfway between the assay baseline and maximum) were calculated.
A representative EC50 assay curve, for compound (Ia-09), is shown in
7
Assay of TLR7 Agonist Activity
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Example 2
This example describes a method for assaying TLR7 agonist activity of the compounds disclosed in this specification.
Engineered human embryonic kidney blue cells (HEK-Blue™ TLR cells; Invivogen) possessing a human TLR7-secreted embryonic alkaline phosphatase (SEAP) reporter transgene were suspended in a non-selective, culture medium (DMEM high-glucose (Invitrogen), supplemented with 10% fetal bovine serum (Sigma)). HEK-Blue™ TLR7 cells were added to each well of a 384-well tissue-culture plate (15,000 cells per well) and incubated 16-18 h at 37° C., 5% CO2. Compounds (100 nl) were dispensed into wells containing the HEK-Blue™ TLR cells and the treated cells were incubated at 37° C., 5% CO2. After 18 h treatment ten microliters of freshly-prepared Quanti-Blue™ reagent (Invivogen) was added to each well, incubated for 30 min (37° C., 5% CO2) and SEAP levels measured using an Envision plate reader (OD=620 nm). The half maximal effective concentration values (EC50; compound concentration which induced a response halfway between the assay baseline and maximum) were calculated.
8
NF-κB Activation Measurement in RAW-Blue Cells
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RAW-Blue™ cells were seeded the day before the experiment at 5 × 105 cells/mL in 48-well plates (500 μL/well).
These cells express a secreted embryonic alkaline phosphatase (SEAP) when NF-κB is activated. NF-κB activation was measured by measuring the alkaline phosphatase produced by RAW-Blue™ cells using the Quanti-Blue™ reagent (InvivoGen), according to the manufacturer’s instructions.
These cells express a secreted embryonic alkaline phosphatase (SEAP) when NF-κB is activated. NF-κB activation was measured by measuring the alkaline phosphatase produced by RAW-Blue™ cells using the Quanti-Blue™ reagent (InvivoGen), according to the manufacturer’s instructions.
9
Assay of TLR7 Agonist Activity
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Example 2
This example describes a method for assaying TLR7 agonist activity of the compounds disclosed in this specification.
Engineered human embryonic kidney blue cells (HEK-Blue™ TLR cells; Invivogen) possessing a human TLR7-secreted embryonic alkaline phosphatase (SEAP) reporter transgene were suspended in a non-selective, culture medium (DMEM high-glucose (Invitrogen), supplemented with 10% fetal bovine serum (Sigma)). HEK-Blue™ TLR7 cells were added to each well of a 384-well tissue-culture plate (15,000 cells per well) and incubated 16-18 h at 37° C., 5% CO2. Compounds (100 nl) were dispensed into wells containing the HEK-Blue™ TLR cells and the treated cells were incubated at 37° C., 5% CO2. After 18 h treatment ten microliters of freshly-prepared Quanti-Blue™ reagent (Invivogen) was added to each well, incubated for 30 min (37° C., 5% CO2) and SEAP levels measured using an Envision plate reader (OD=620 nm). The half maximal effective concentration values (EC50; compound concentration which induced a response halfway between the assay baseline and maximum) were calculated.
10
NF-κB Activation by Fungal PAMPs
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Raw-Blue cells (Invivogen) is derived from mouse macrophage RAW 264.7 cell line with chromosomal integrated a secreted embryonic alkaline phosphatase (SEAP) reporter construct inducible by NF-κB expression. 5 × 104 Raw-Blue cells were seeded with blocking antibodies against each pattern recognition receptor (used at 25 μg/mL each) 30 min prior to fungal β-1,3-glucan (10 μg/mL) or mannan (500 μg/mL) stimulation. Supernatants was collected 16 h after stimulation, and SEAP secretion reflective of NF-κB activation was detected by colorimetric assay using Quanti-Blue reagent (Invivogen).
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