RAW-Blue™ mouse macrophage reporter cells (Invivogen) were cultured to measure TLR-mediated immune stimulation following the manufacturer’s protocol. Briefly, cells (5000 cells in 100 μL in a 96-well dish) were culture overnight and then treated with PBS (1 μl), CpG (1 μl of 1 μg/ul stock) SWNT/LP (1 μl of 1 μg/ul stock ) or SWNT/CpG (1 μl of 1 μg/ul stock ) for 16 hours. The level of secreted embryonic alkaline phosphatase (SEAP) was quantified using QuantiBlue™ substrate (InvivoGen) and measured after 2 hours at 620 nm absorption using DTX 880 Multimode Detector (Beckman Coulter). QUANTI-Blue substrate was prepared according to manufacturer’s protocol (InvivoGen).
Quanti blue substrate
Manufactured by InvivoGen
Sourced in United Kingdom
Quanti-Blue substrate is a colorimetric detection reagent used to quantify alkaline phosphatase (AP) activity. It undergoes a color change upon cleavage by AP, which can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Epoch elisa reader, by Agilent Technologies (6 mentions)
96 well tissue culture plate, by Thermo Fisher Scientific (6 mentions)
Mst14, by R&D Systems (6 mentions)
Hek blue ifna b cells, by InvivoGen (3 mentions)
Recombinant ifnα, by Novus Biologicals (3 mentions)
Hek blue ifnα b cells, by InvivoGen (3 mentions)
Recombinant ifna, by Novus Biologicals (3 mentions)
Dtx 880 multimode detector, by Beckman Coulter (2 mentions)
Raw blue mouse macrophage reporter cells, by InvivoGen (2 mentions)
Raw blue cells, by InvivoGen (2 mentions)
Zeocin antibiotic, by InvivoGen (2 mentions)
Zeocin, by InvivoGen (2 mentions)
Raw blue macrophages, by InvivoGen (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Vitamin d3, by Merck Group (1 mentions)
Quanti luc substrate, by InvivoGen (1 mentions)
Infinite f200 pro plate reader, by Tecan (1 mentions)
Hek blue ifn λ cells, by InvivoGen (1 mentions)
Hek blue ifn α β, by InvivoGen (1 mentions)
Envision plate reader, by PerkinElmer (1 mentions)
29 protocols using quanti blue substrate
1
Quantifying TLR-mediated Immune Stimulation
2
Measuring TLR-Mediated Immune Stimulation
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RAW-Blue™ mouse macrophage reporter cells (Invivogen) were cultured to measure TLR-mediated immune stimulation following the manufacturer’s protocol. Briefly, cells (5000 cells in 100 μL in a 96-well dish) were treated with 1 μL of PBS, RSS-CpG (1μg/μl) or SWCNT/CpG-2 (1μg/μl) for 16 hours. RAW-Blue™ cells induce the activation of NF-κB and AP-1, and subsequently the secretion of quantifiable secreted embryonic alkaline phosphatase (SEAP). The level of secreted embryonic alkaline phosphatase (SEAP) was quantified using QuantiBlue™ substrate (InvivoGen) and measured at 620 nm absorption using DTX 880 Multimode Detector (Beckman Coulter). QUANTI-Blue substrate was prepared according to manufacturer’s protocol (InvivoGen).
3
THP-1 Cell Line Activation Assay
4
Masking Effect on IFNα Activity
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Example 21
In this example, it is shown that both Mask1 and Mask2 of the disclosure can reduce and restore IFNα activity. Briefly, HEK Blue IFNa/b cells (Invivogen) were seeded into 96-well tissue culture plates (Fisher) at a density of 1×10e4 cells/well (50 uL/well). 50 uL/well of recombinant IFNα (Novus Biologicals) or indicated Abs (5T4 or Mesothelin) were incubated with the cells at the indicated concentrations overnight at 37 deg. C. Abs cleaved with MST14 (R&D Systems) were prepared by incubating 50 ug of Ab with 0.5 ug of MST14 for 1 hr. At 37 deg. C. Then, 10 uL of supernatant was added to a plate containing 90 uL/well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader. The results show that the IFNα activity in masked anti-CD138-IFNα (Mask1) and masked anti-CD138-IFNα (Mask2) was reduced compared to when the mask is cleaved. (See,
5
Reduction of IFNα Activity by Masked Fusion Abs
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Example 6
In this example, it is shown that a plurality of Masked Fusion Abs of the disclosure can reduce and restore IFNα activity. Briefly, HEK Blue IFNa/b cells (Invivogen) were seeded into 96-well tissue culture plates (Fisher) at a density of 1×10e4 cells/well (50 uL/well). 50 uL/well of recombinant IFNα (Novus Biologicals) or indicated Abs (5T4 or Mesothelin) were incubated with the cells at the indicated concentrations overnight at 37 deg. C. Abs cleaved with MST14 (R&D Systems) were prepared by incubating 50 ug of Ab with 0.5 ug of MST14 for 1 hr. At 37 deg. C. Then, 10 uL of supernatant was added to a plate containing 90 uL/well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader. The results show that the IFNα activity in masked anti-5T4-IFNα and masked anti-mesothelin-IFNα was reduced by approximately 1 to 2 logs compared to when the mask is cleaved. (See,
6
Masking of IFNα Activity by Fusion Abs
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Example 6
In this example, it is shown that a plurality of Masked Fusion Abs of the disclosure can reduce and restore IFNα activity. Briefly, HEK Blue IFNα/b cells (Invivogen) were seeded into 96-well tissue culture plates (Fisher) at a density of 1×10e4 cells/well (50 uL/well). 50 uL/well of recombinant IFNa (Novus Biologicals) or indicated Abs (5T4 or Mesothelin) were incubated with the cells at the indicated concentrations overnight at 37 deg. C. Abs cleaved with MST14 (R&D Systems) were prepared by incubating 50 ug of Ab with 0.5 ug of MST14 for 1 hr. At 37 deg. C. Then, 10 uL of supernatant was added to a plate containing 90 uL/well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader. The results show that the IFNα activity in masked anti-5T4-IFNα and masked anti-mesothelin-IFNα was reduced by approximately 1 to 2 logs compared to when the mask is cleaved. (See,
7
NF-κB/AP1 Reporter Assay in THP-1 Cells
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THP1-Blue and THP1-Blue-CD14 cells are derived from the myelomonocytic THP-1 cell line and contain a nuclear factor kappa B (NF-κB)/activator protein 1 (AP1) inducible reporter construct for secreted embryonic alkaline phosphatase (SEAP). The latter also overexpresses CD14 for enhanced sensitivity toward toll-like receptor (TLR) stimulation.
Upon stimulation with LPS, SEAP expression was initiated. SEAP protein is secreted into the supernatant, and its activity can be measured colorimetrically at 635 nm by adding 10 µL cell supernatant to 100 µL QUANTI-Blue substrate (InvivoGen, San Diego, CA, USA). After an incubation period of 70 min at 37 °C protected from light, SEAP activity was determined at 625 nm using a Tecan infinite F200 PRO plate reader (Männedorf, Switzerland).
Upon stimulation with LPS, SEAP expression was initiated. SEAP protein is secreted into the supernatant, and its activity can be measured colorimetrically at 635 nm by adding 10 µL cell supernatant to 100 µL QUANTI-Blue substrate (InvivoGen, San Diego, CA, USA). After an incubation period of 70 min at 37 °C protected from light, SEAP activity was determined at 625 nm using a Tecan infinite F200 PRO plate reader (Männedorf, Switzerland).
8
Quantitative IFN Secretion Assay
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HEK-Blue IFN-α/β and HEK-Blue IFN-λ cells (InvivoGen) were plated at 30,000 cells per well in a 96-well plate. The following day, medium from infected cells (or control cells) was added and a standard curve was generated in parallel by serial dilutions of type I or type III IFNs in complete DMEM. After 20 to 24 h of incubation, 30 μl of HEK-Blue IFN-α/β supernatants was added to 120 μl of Quanti-blue substrate (InvivoGen) and incubated at 37°C for 15 min. Absorbance was measured at 620 nm using an Envision plate reader (Perkin-Elmer). The standard curves were used to provide semiquantitative analyses of the IFN concentrations produced by the infected cells.
9
Bioactivity Assay for IL-33 and IL-1β
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HEK-Blue IL-33/IL-1β (HEK293-ST2L) cells were purchased from Invivogen (Toulouse, France) and cultivated according to the manufacturer's protocol in DMEM (Greiner bio-one, Frickenhausen, Germany) with 5% FCS (GE Healthcare, IL, USA), 2 mM l-glutamine, 100 IU/ml penicillin and 100 IU/ml streptomycin at 5% CO2, 37°C. 30 μg/ml blasticidin, 200 μg/ml hygromycin B Gold and 100 μg/ml zeocin were added after the second passage to maintain the plasmids encoding IL1R1, co-receptor IL-1RAcP, ST2L, and the gene for SEAP. To analyze IL-33 or IL-1β bioactivity, 5 × 104 HEK293-ST2L cells were resuspended in 180 μl medium and were co-incubated with 20 μl of sample. In some experiments, 5 × 104 HEK293-ST2L cells were resuspended in 160 μl and co-incubated with 20 μl sample and 20 μl serum or plasma for a total final volume of 200 μl. For inhibition of proteases, protease inhibitor cocktail (Sigma-Aldrich, Steinhein, Germany) was added in a dilution of 1:160 directly to the cell suspension. After a 22 h incubation period, 40 μl of medium supernatant were co-incubated with 160 μl QUANTI-blue substrate (Invivogen, Toulouse, France) and SEAP activity was measured photometrically at 635 nm for 2 h every minute. Cell viability of HEK293-ST2L cells was assessed by MTT assay to exclude cytotoxic effects mediated by the samples (Roche Life Sciences, Basel, Switzerland).
10
Quantifying Smad2/3 Activation by TGF-β
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Activation of Smad2/3 signals by TGF-β family members including TGF-β1, 2, 3, and Activin A was determined using reporter assay which employs HEK-BlueTM TGF-β cells allowing direct quantification of soluble alkaline phosphatase (SEAP) expressed under control of Smad3/4-inducible elements. HDLECs were treated with TGF-β2 or SB431542 for 48 h. The culture medium was then replaced with EGM-2MV medium with 0.5% FBS and HDLECs were incubated for additional 6 h. The conditioned medium was then collected and added to the pre-seeded HEK-BlueTM TGF-β reporter cells, followed by incubation for 24 h. Fraction of the medium obtained from incubation with HEK-Blue cells was then mixed with QUANTI-Blue substrate (InvivoGen) and incubated for 30 min. The released SEAP was quantified at 640 nm using spectrophotometer.
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