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Raw blue nf κb cells

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RAW-Blue NF-κB cells are a reporter cell line derived from the RAW 264.7 mouse macrophage cell line. These cells stably express a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a NF-κB-inducible promoter. The SEAP activity in the cell culture supernatant can be measured to monitor NF-κB activation.

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Lab products found in correlation

Multiskan fc plate reader, by Thermo Fisher Scientific (4 mentions) Quanti blue, by InvivoGen (4 mentions)

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RAW-Blue cells (71 citations) RAW-Blue (17 citations)

4 protocols using raw blue nf κb cells

1

NF-κB Activation Assay for MPs

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RAW-Blue NF-κB cells (Invivogen) were passaged and plated in a 96 well plate at 100k cells/well in 180 μL DMEM containing 10% HIFBS. Cells were incubated at 37°C and 5% CO2 for 24 h. 100 ul of cells were incubated with varying ratios of MPs at 37°C and 5% CO2 for 18 h. After 18 h, 20 μL of the cell supernatant was placed in 180 μL freshly prepared QuantiBlue (Invivogen) solution and incubated at 37°C/5% CO2 for up to 2 h. The plate was analyzed every hour using a Multiskan FC plate reader (Thermo Scientific) and absorbance was measured at 620 nm BCA Assay- This was performed according to manufacturer’s instruction (Thermo Fischer) with some modifications. 100 million beads were incubated with BCA solution and reacted for 30 mins at 60°C then analyzed every hour using a Multiskan FC plate reader (Thermo Scientific) and absorbance was measured at 562 nm and compared to a standard curve of modified MPLA or Pam2 after subtracting a background of maleimide modified MP.
Kong D, & DePamphilis M.L. (2001). Site-Specific DNA Binding of the Schizosaccharomyces pombe Origin Recognition Complex Is Determined by the Orc4 Subunit. Molecular and Cellular Biology, 21(23), 8095-8103.
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2

NF-κB Activation Assessment in Cells

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RAW-Blue NF-κB cells (Invivogen) were challenged similarly to BMDCs as above. After 18 h of second TLR agonist challenge, 20 μL of the cell supernatant was placed in 180 μL freshly prepared QuantiBlue (Invivogen) solution and incubated at 37 °C/5% CO2 for up to 2 h. The plate was analyzed every hour using a Multiskan FC plate reader (Thermo Scientific) and absorbance was measured at 620 nm.
Yamamoto N, & Homma S. (1991). Vitamin D3 binding protein (group-specific component) is a precursor for the macrophage-activating signal factor from lysophosphatidylcholine-treated lymphocytes.. Proceedings of the National Academy of Sciences of the United States of America, 88(19), 8539-8543.
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3

NF-κB Cell Activation Assay

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RAW-Blue NF-κB cells (Invivogen) were passaged and plated in a 96 well plate at 100k cells/well in 180 μL DMEM containing 10% HIFBS. Cells were incubated at 37 °C and 5% CO2 for 1 h. Particles were counted using flow cytometer. 100k particles were added to each well (1 particle:1 cell). The volume of each well was brought to 200 μL and incubated at 37 °C and 5% CO2 for 18 h. After 18 h, 20 μL of the cell supernatant was placed in 180 μL freshly prepared QuantiBlue (Invivogen) solution and incubated at 37 °C/5% CO2 for up to 2 h. The plate was analyzed every hour using a Multiskan FC plate reader (Thermo Scientific) and absorbance was measured at 620 nm.
Moser B.A., Steinhardt R.C, & Esser-Kahn A.P. (2016). Surface Coating of Nanoparticles Reduces Background Inflammatory Activity while Increasing Particle Uptake and Delivery. ACS biomaterials science & engineering, 3(2), 206-213.
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4

NF-κB Activation Assay for MPs

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RAW-Blue NF-κB cells (Invivogen) were passaged and plated in a 96 well plate at 100k cells/well in 180 μL DMEM containing 10% HIFBS. Cells were incubated at 37°C and 5% CO2 for 24 h. 100 ul of cells were incubated with varying ratios of MPs at 37°C and 5% CO2 for 18 h. After 18 h, 20 μL of the cell supernatant was placed in 180 μL freshly prepared QuantiBlue (Invivogen) solution and incubated at 37°C/5% CO2 for up to 2 h. The plate was analyzed every hour using a Multiskan FC plate reader (Thermo Scientific) and absorbance was measured at 620 nm BCA Assay- This was performed according to manufacturer’s instruction (Thermo Fischer) with some modifications. 100 million beads were incubated with BCA solution and reacted for 30 mins at 60°C then analyzed every hour using a Multiskan FC plate reader (Thermo Scientific) and absorbance was measured at 562 nm and compared to a standard curve of modified MPLA or Pam2 after subtracting a background of maleimide modified MP.
Deak P., Studnitzer B., Ung T., Steinhardt R., Swartz M, & Esser-Kahn A. (2022). Isolating and targeting a highly active, stochastic dendritic cell subpopulation for improved immune responses. Cell reports, 41(5), 111563.
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