pSEAP-TonE was a gift from Prof. W. Neuhofer [17 (link)]. Briefly, two TonEBP responsive elements were cloned upstream of the secreted human placental alkaline phosphatase (SEAP) coding region, in the pSEAP2-Basic vector (GenBank Accession # U89937 ) from Clontech (Mountain View, CA). Cells were transfected transiently as described above. After 24 h, cells were treated for 24 h either in iso-osmolar or hyperosmolar (additional 100 mM NaCl) medium. The cell culture medium supernatant was collected, cleared with centrifugation, and stored at −20 °C until assayed. SEAP activity in the cell culture medium was assayed using the Phospha-Light™ SEAP Reporter Gene Assay System (Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions, where 120 µl of culture medium was diluted in an equal volume of kit 1X dilution buffer. After treatment as described in the user’s manual, samples were assayed in duplicates of 50 µl. Samples were measured in a Wallac Victor2 Luminometer Multilabel Counter (model 1420–042b, PerkinElmer, Waltham, MA). Luminescent results were calculated from a standard curve (ranging from 0 to 5 mU/ml of standard SEAP activity) of standard SEAP supplied in the Phospha-Light™ SEAP Reporter Gene Assay System. Results were then expressed as the relative modulation compared to the control iso-osmolar condition.
Phospha light seap reporter gene assay system
Manufactured by Thermo Fisher Scientific
The Phospha-Light™ SEAP Reporter Gene Assay System is a chemiluminescent reporter gene assay used to quantify the activity of secreted alkaline phosphatase (SEAP) in cell culture supernatants. The assay provides a sensitive and convenient method for monitoring gene expression and cellular activity.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pseap2 basic vector, by Takara Bio (1 mentions)
Gaussia luciferase assay kit, by New England Biolabs (1 mentions)
Synergy htx microplate reader, by Agilent Technologies (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Penicillin streptomycin, by American Type Culture Collection (1 mentions)
Fugene 6, by Promega (1 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
Clariostar plus, by BMG Labtech (1 mentions)
Tristar2 lb 942, by Berthold Technologies (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
7 protocols using phospha light seap reporter gene assay system
1
TonEBP-Responsive SEAP Assay
2
Luciferase Assay for Monitoring MondoA Activity
3
Quantifying SEAP Activity in BocK-Treated HEK293 Cells
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HEK293 cells were cultured in 24-well plates in DMEM, with Glutamine XL (Quality Biological), containing 10% FBS (Sigma Aldrich) and penicillin-streptomycin (American Type Culture Collection). Transfections were performed using FuGene 6 (Promega) and 0.5 µg total DNA, with a 6:1 FuGene:DNA ratio. After 4 h, the media was replaced with fresh media containing 2 mM BocK, ABA (10, 200, or 500 µM), or vehicle control (15% DMSO/0.2 M NaOH), and the cells were cultured at 37 °C (5% CO2) for 2 d. A sample of the media from each well was taken for the SEAP activity assay (Phospha-Light™ SEAP Reporter Gene Assay System, Applied Biosystems), and the assay was performed according to the manufacturer’s protocol. Luminescence was measured (1 s/well) using a Synergy HTX microplate reader (BioTek) and graphed using GraphPad Prism. Data are displayed as the mean ± SEM of four biological replicates. Data were normalized by setting the condition that achieved maximum signal equal to 100%.
4
SEAP Reporter Assay for Transfected HEK293T
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For transfection on HEK293T cells, pSEAP vector was used to co-transfect HEK293T cells with vectors expressing AnkX, SseKs or their catalytic mutants following the transfection method described above. 16 h post transfection or infection, 200 μl DMEM GlutaMAX (Gibco) media was added to each well to replace old media and the 24 well plate was put back at 37°C with 5% CO2 for a further 8 h incubation. 24 h post transfection or infection, supernatants and cells lysates were collected for processing using Phospha-LightTM SEAP Reporter Gene Assay System (ThermoFisher Scientific) following manufacturer's instructions. Processed samples were plated on a 96-well white flat bottom plate and read via CLARIOstar Plus (BMG LABTECH).
5
Quantifying Secreted Alkaline Phosphatase
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A total of 4.8×10 6 pancreatic β-cells were transfected with a plasmid carrying secretory alkaline phosphatase (SEAP, kindly provided by Dr. Paul Randazzo, National Cancer Institute, Bethesda, MD, USA) with Lipofectamine 2000 by reverse transfection, following the manufacturer's protocol (Thermo Fisher Scientific). They were plated on a 6-well plate. Two days after transfection, the cells were washed twice and incubated with 1 ml of culture medium for times indicated in Fig. 1F at 37°C with either DMSO or 4μ8C. Then, 50 μl of culture medium was collected to determine the amount of secreted SEAP. The cells were washed twice with PBS and lysed in 200 μl of lysis buffer (0.1% Triton X-100, 1× dilution buffer). Then, 10 μl of lysate was obtained to determine the amount of SEAP in the cell lysate. The medium and the lysate were processed as per the manufacturer ' s protocol (Phospha-Light TM SEAP Reporter Gene Assay System; Thermo Fisher Scientific). Chemiluminescence was measured using the TriStar2 LB 942 (Berthold, Bad Wilbad, Germany). SEAP secretion was calculated by dividing the amount of SEAP in the medium by the total amount of SEAP in the medium and the lysate.
6
SREBP-2 Processing Assay Using PLAP
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Analysis of SREBP-2 processing was measured by a secreted alkaline phosphatase assay as described previously35 (link). In brief, HEK-293T expressing EGFP-NBEAL1 cells were seeded in 24-well plates at 1.2 × 105 cells per well and incubated for 24 h. Cells were co-transfected with 0.05 µg pRL-CMV and 0.45 µg pCMV-PLAP-BP2 using Lipofectamine 2000. The pCMV- PLAP-BP2 contains a fusion protein, which codes for placental alkaline phosphatase (PLAP, amino acid 1–506) and the regulatory domain of SREBP2 (BP2, amino acid 513–1141). The PLAP is flanked by a cleavage sites for a signal peptidase and Site-1 protease (S1P) and cleaved by endogenous S1P upon SREBP2 processing. When SREBP2 is processed, the cleavage of both proteases leads to PLAP secretion into the media. PLAP was measured by Phospha-Light™ SEAP Reporter Gene Assay System according to manufacturer’s recommendation (Thermo Fisher Scientific). The cells were immediately lysed in passive lysis buffer (Promega). The transfection level of pRL-CMV was detected using the Stop-Glow component from Dual-Luciferase® Reporter Assay System (Promega) according to manufacturer’s protocol and used as a control for transfected cells.
7
AAV-mediated SEAP Reporter Assay
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For viral transduction of siRNA treated cells, 24 h after siRNA transfection HEK-293 cells were transduced with AAV1-mSEAP at a multiplicity of infection (MOI) of 5,000 viral genomes per cell. The medium was replaced by fresh medium after 16 hours of AAV incubation. Twenty four hours later, cell supernatants were collected for Alkaline phosphatase activity measurements and cells were harvested for quantifications of mSEAP mRNA and vector genome contents.
Quantification of alkaline phosphatase activity was performed with the Phospha-Light SEAP Reporter Gene Assay System (ThermoFisher) according to manufacturer instructions. Chemiluminescence was measured in microplates in a luminometer (FlexStation 3). Raw data were analyzed through simple spreadsheet and quantified by using standard curve previously prepared by serially diluting stock enzyme.
Quantification of alkaline phosphatase activity was performed with the Phospha-Light SEAP Reporter Gene Assay System (ThermoFisher) according to manufacturer instructions. Chemiluminescence was measured in microplates in a luminometer (FlexStation 3). Raw data were analyzed through simple spreadsheet and quantified by using standard curve previously prepared by serially diluting stock enzyme.
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