Blood was collected through cardiac puncture, and serum was stored at −80°C until use in the RAW‐Blue assay. PAMPs were assayed with RAW‐Blue™ cells (InvivoGen, San Diego, CA) through detection of NF‐κB/AP‐1 activation following activation of TLRs (with the exception of TLR5), NOD1/2, and dectin‐1 using a modified version of the manufacturer's protocol. RAW‐Blue™ cells were grown in growth medium (DMEM, 4.5 g/l glucose, 2 mM L‐glutamine, 10% fetal bovine serum (FBS), 100 μg/ml Zeocin™ (InvivoGen, San Diego, CA)). The assay was performed when the cells were in passage 10–15 by plating 105 cells in 96‐well plates containing basal DMEM. After 6 h of starvation 30 μl of mouse serum, 30 μl of FBS(−control) or 30 μl of FBS+LPS (+ control) was added per well and cells were incubated for 21 h at 37° C under an atmosphere of 5% CO2/95% air. SEAP levels were determined using a spectrophotometer at 620–655 nm after a 1‐ to 3‐h incubation at 37° C of 20 μl of induced RAW‐Blue™ cell supernatant with 180 μl QUANTI‐Blue™ (InvivoGen, San Diego, CA).
Raw blue cells
Manufactured by InvivoGen
Sourced in United States, France
RAW-Blue cells are a reporter cell line derived from the RAW 264.7 murine macrophage cell line. They stably express a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a NF-κB-inducible promoter. These cells can be used to monitor NF-κB activation in response to various stimuli.
Automatically generated - may contain errors
Lab products found in correlation
Quanti blue, by InvivoGen (12 mentions)
Quanti blue reagent, by InvivoGen (10 mentions)
Zeocin, by InvivoGen (7 mentions)
Quanti blue assay, by InvivoGen (7 mentions)
Normocin, by InvivoGen (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Raw blue cells, by Corning (3 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Corning (2 mentions)
Fetal bovine serum (fbs), by Lonza (2 mentions)
M200 microplate reader, by Tecan (2 mentions)
Dulbecco s modified eagle s medium dmem, by Corning (2 mentions)
Penicillin streptomycin, by InvivoGen (2 mentions)
Thp1 xblue cells, by InvivoGen (2 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (2 mentions)
Normicin, by InvivoGen (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
71 protocols using raw blue cells
1
Serum PAMP Activation Assay
2
Measuring NF-κB Activity in RAW-Blue Cells
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 NF-κB Activation in RAW-Blue Cells
5
Culturing HEK293T-hACE2 and Cell Lines
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HEK293T cells expressing human angiotensin I-converting enzyme 2 (HEK293T-hACE2) were kindly provided by Jesse Bloom (Fred Hutchinson Cancer Research Center, Seattle, WA, USA) (46 (link)). The Lenti-X 293T cell line was purchased from TaKaRa Bio USA, Inc. (San Jose, CA). RAW-Blue cells were purchased from Invivogen (San Diego, CA, USA). These cell lines were maintained in complete Dulbecco’s modified Eagle’s medium (DMEM) (Corning, Corning, NY) supplemented with 10% fetal bovine serum (FBS) (Corning) and 100 U/mL penicillin-streptomycin (Corning) at 37°C in a humidified incubator with 5% CO2. Cells at passages 2 to 10 were used for the experiments.
6
Multimodal Immunological Assays for EAE
7
Regulation of IL-1β and COX-2 Expression
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RAW Blue cells were purchased from InvivoGen (San Diego, CA), used at passages under 15, and cultured in DMEM growth medium supplemented with 10% serum, 50 U/mL penicillin and 200 mg/mL zeocin. Cells were grown in regular conditions (37°C, 5% CO2), serum starved overnight, and treated with 100 ng/mL IL-1β for 4 h. Cells were, respectively pre-incubated for 30 min with peptides 1 or 2 (10−6 M) or Kineret (1.0 mg/mL) to reach equilibrium prior to the experiments (n = 4 each treatment). Cells were harvested and incubated for 5 min in RIBOzol (AMRESCO). RNA was extracted according to manufacturer's protocol and RNA concentration and integrity were measured with a NanoDrop 1,000 spectrophotometer. A total of 500 ng of RNA was used to synthesize cDNA using iScript Reverse Transcription SuperMix (Bio-Rad, Hercules, CA). Primers (Table 1 ) were designed using National Center for Biotechnology Information Primer Blast. Quantitative gene expression analysis was performed using the Stratagene MXPro3000 (Stratagene) with SYBR Green Master Mix (Bio-Rad). Gene expression levels were normalized to 18S universal primer (Ambion Life Technology, Burlington ON, Canada). Genes analyzed include IL1β and PTGHS2 [Prostaglandin H synthetase 2 or cyclooxygenase-2 (COX-2)]. Data are representative of 3 experiments (each with n = 4 per treatment group).
8
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).
9
Dex P-A Hydrogel Modulates Immune Response
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200 μL of the
DexP-A hydrogel unloaded or loaded with 10 μM
SIINFEKL was added per well to a 24-well plate. 500,000 D1 cells were
seeded per well in a final volume of 300 μL of DMEM medium.
A transwell insert of 0.45 microns (corning ref. 354,572) was placed
per well on the plate. Within the transwell insert, 100,000 RAW-Blue
cells (Invivogen, raw-sp) were seeded in 200 μL of DMEM. As
a positive control of activation, 1 μg mL–1 LPS-EK was added to the RAW-Blue cells. Additionally, we prepared
cocultures of D1 and RAW-blue cells using transwell inserts without
the DexP-A hydrogel. After 24 h incubation at 37 °C,
NF-κB/AP-1 activation upon the previously mentioned stimuli
was assessed by measuring the release of secreted embryonic alkaline
phosphates (SEAPs) by the RAW-Blue cells. For this purpose, 20 μL
of the induced RAW-Blue cell supernatant was added to 180 μL
of QUANTI-Blue solution (Invivogen rep-qbs) in a 96-well plate. The
samples were incubated for 3 h at 37 °C. SEAP release was measured
at 655 nm.
Dex
SIINFEKL was added per well to a 24-well plate. 500,000 D1 cells were
seeded per well in a final volume of 300 μL of DMEM medium.
A transwell insert of 0.45 microns (corning ref. 354,572) was placed
per well on the plate. Within the transwell insert, 100,000 RAW-Blue
cells (Invivogen, raw-sp) were seeded in 200 μL of DMEM. As
a positive control of activation, 1 μg mL–1 LPS-EK was added to the RAW-Blue cells. Additionally, we prepared
cocultures of D1 and RAW-blue cells using transwell inserts without
the Dex
NF-κB/AP-1 activation upon the previously mentioned stimuli
was assessed by measuring the release of secreted embryonic alkaline
phosphates (SEAPs) by the RAW-Blue cells. For this purpose, 20 μL
of the induced RAW-Blue cell supernatant was added to 180 μL
of QUANTI-Blue solution (Invivogen rep-qbs) in a 96-well plate. The
samples were incubated for 3 h at 37 °C. SEAP release was measured
at 655 nm.
10
Evaluating BLP Activation of NF-κB
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The capacity of BLP to activate NF-κB via Toll-like receptor-2 was evaluated using RAW-Blue™ cells (InvivoGen, Toulouse, France). RAW-Blue™ cells have a number of pattern recognition receptors which when bound to agonists leads to activation of NF-κB and thus the production of secreted alkaline phosphatase. A schematic illustration is shown in Fig. S1 . Cells were maintained in DMEM with high glucose (Gibco Life Technologies BV, Bleiswijk, The Netherlands), 10% FBS (Lonza, Basel, Switzerland), 100 µg/ml Normocin™ (InvivoGen, Toulouse, France), 2 mM L-glutamine and passaged when 70–80% confluency was reached. Approximately 5 × 104 cells were added to 96-well flat bottom plates and were stimulated with 1.7 µg untreated BLP or with liquid and reconstituted SFD powder formulations with (5 µg HA + 300 µg BLP formulation) or without 1.7 µg BLP. The incubation was maintained for 18 h at 37 °C with 5% CO2. To measure alkaline phosphatase levels, 150 µL QUANTI-Blue™ (InvivoGen, Toulouse, France) was added to the cell supernatant and after 1 h, absorbance was measured at 630 nm using a spectrophotometer.
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