Seap reporter assay kit

Manufactured by InvivoGen

Sourced in United States

The SEAP Reporter Assay Kit is a laboratory reagent designed to measure secreted embryonic alkaline phosphatase (SEAP) activity in cell culture supernatants. The kit provides the necessary components to quantify SEAP levels, which can be used as a reporter for gene expression or cell signaling studies.

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Lab products found in correlation

Biolux gaussia luciferase assay kit, by New England Biolabs (2 mentions) Radioimmunoprecipitation assay lysis buffer, by Thermo Fisher Scientific (1 mentions) Quant microplate reader, by Agilent Technologies (1 mentions)

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SEAP) reporter (3 citations)

5 protocols using seap reporter assay kit

Evaluation of GSDMC Promoter Activity

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Cells were transfected with GSDMC promoter luciferase reporter plasmids. After transfection for 48 h, cells were collected and detected using BioLux Gaussia Luciferase Assay Kit (#E3300, New England BioLabs) and SEAP Reporter Assay Kit (#rep-sap, InvivoGen) following the manufacturers’ protocols.

Hou J., Zhao R., Xia W., Chang C.W., You Y., Hsu J.M., Nie L., Chen Y., Wang Y.C., Liu C., Wang W.J., Wu Y., Ke B., Hsu J.L., Huang K., Ye Z., Yang Y., Xia X., Li Y., Li C.W., Shao B., Tainer J.A, & Hung M.C. (2020). PD-L1-Mediated Gasdermin C Expression Switches Apoptosis to Pyroptosis in Cancer Cells and Facilitates Tumor Necrosis. Nature cell biology, 22(10), 1264-1275.

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Evaluation of GSDMC Promoter Activity

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SEAP Reporter Assay in HEK293T Cells

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SEAP assay was performed by an SEAP reporter assay kit (Invivogen). HEK293T cells were cultured in 96-well plates at a density of 1 × 10⁴ cells per well and transfected with the LegA15 expression construct. Its empty vector was used as a control. The cells and culture medium were separated at 24 hours of transfection by a centrifugation at 10,000 rpm for 5 min. The culture medium supernatant was transferred to a new tube and heated at 65°C for 10 min to inhibit endogenous alkaline phosphatases. The cells were lysed by radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific) on ice for 30 min and centrifuged at 10,000 rpm for 5 min. In addition, their supernatant was collected into a new tube and also heated at 65°C for 10 min. The SEAP activity was detected at 410 nm according to the manufacturer’s manual and repeated at least three times.

Chen T.T., Lin Y., Zhang S., Liu S., Song L., Zhong W., Luo Z.Q, & Han A. (2022). Atypical Legionella GTPase effector hijacks host vesicular transport factor p115 to regulate host lipid droplet. Science Advances, 8(50), eadd7945.

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Quantifying Secreted Alkaline Phosphatase

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Cells were transfected with indicated amounts of pCE-secreted alkaline phosphatase (pCE-SEAP) plasmid and FuGENE6 reagent. The following day, medium was changed, and after two more days, culture medium was collected and separated from cellular debris by centrifugation. Supernatants were heated to 65 °C in order to inhibit endogenous alkaline phosphatase. To determine enzyme activity, a SEAP Reporter Assay Kit (InvivoGen) was used. Briefly, 10 μl of cell culture medium was mixed with 25 μl of 1× dilution buffer, 50 μl of 1× assay buffer, 10 μl of 100 mM L-homoarginine, and 20 μl of staining solution (p-nitrophenyl phosphate, 75 μM). Samples were incubated at 37 °C for 1 h before the absorbance was measured at 450 nm.

Drozd A.M., Walczak M.P., Piaskowski S., Stoczynska-Fidelus E., Rieske P, & Grzela D.P. (2015). Generation of human iPSCs from cells of fibroblastic and epithelial origin by means of the oriP/EBNA-1 episomal reprogramming system. Stem Cell Research & Therapy, 6(1), 122.

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Transient Transfection of HEK293 Cells

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HEK293 cells (5 × 10⁶) were transiently transfected with 5.7 µg of ELAM-SEAP reporter gene (Invivogen, San Diego, CA, USA) and 0.3 µg of TLR2/TLR6 expression vector (Invivogen, CA) using the FuGENE 6 transfection kit (Roche Diagnostics, Mannheim, Germany). The cells were seeded into 96-well plates 6 h after transfection. After 24 h, the cells were treated with lipopeptides (1 µM) and the medium was collected 6 h after stimulation. SEAP activity was measure using SEAP reporter assay kit (Invivogen, CA) and µQuant Microplate Reader (BioTek, Winooski, VT, USA) at OD₄₁₂.

Zhou Y., Banday A.H., Hruby V.J, & Cai M. (2019). Development of N-Acetylated Dipalmitoyl-S-Glyceryl Cysteine Analogs as Efficient TLR2/TLR6 Agonists. Molecules, 24(19), 3512.

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