RAW-Blue™ cells were first incubated for 30 min with or without PDTC (30 mM), sc-3060 (0.1–3 μM) or NAC (10 mM) and then incubated for 24 h with or without CGL (1–10 μg/ml). In the LPS tolerance assay, RAW-Blue™ cells were incubated for 24 h with or without CGL (1–10 μg/ml) or LPS (0.1 μg/ml) and then changed to fresh medium and incubated for 24 h with or without LPS (1 μg/ml). The medium (20 μl) from the treated RAW-Blue™ cells was mixed with 200 μl of QUANTI-Blue™ medium (Invitrogen, Carlsbad, CA) in 96-well plates and incubated at 37 °C for 15 min. Secreted embryonic alkaline phosphatase activity was assessed by measuring the optical density at 655 nm using a microplate absorbance reader.
Quanti blue medium
Manufactured by Thermo Fisher Scientific
QUANTI-Blue™ medium is a colorimetric detection medium for quantifying alkaline phosphatase (AP) activity. It is designed for the detection and quantification of AP in biological samples.
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Lab products found in correlation
3 protocols using quanti blue medium
1
Modulation of RAW-Blue Cell Activation
2
Measuring NF-κB Activation in J-Blue Cells
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NF-κB reporter cells (J-Blue cells) were derived from J774A.1 macrophages with chromosomal integration of a secreted embryonic alkaline phosphatase (SEAP) reporter plasmid (pNiFty2-SEAP from InvivoGen, Carlsbad, CA). J-Blue cells were incubated for 30 min with or without NAC (10 mM) and then infected for 24 h with or without NG. The medium (20 μl) from the infected J-Blue cells was mixed with 200 μl QUANTI-Blue medium (Invitrogen) in 96-well-plates and incubated for 15 min at 37°C. SEAP activity was assessed by measuring the optical density at 655 nm using a microplate absorbance reader.
3
Quantification of NF-κB-Induced SEAP in J-Blue Cells
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J-Blue cells, J774A.1 macrophages stably expressing the gene for secreted embryonic alkaline phosphatase induced by NF-κB, were seeded in a 24-well plate at a density of 2 × 105 cells in 0.5 mL medium and grown overnight in a 5% CO2 incubator at 37 oC. Cells were then treated with negative control, positive control, or ACP and incubated for another 24 h. The medium (20 μL) from treated J-Blue cells was mixed with 200 μL of QUANTI-Blue™ medium (Invitrogen, Carlsbad, CA) in 96-well plates and incubated at 37 °C for 15 min. The secreted embryonic alkaline phosphatase activity was assessed with a microplate absorbance reader measuring the optical density at 655 nm.
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