6 × 105 cells ml−1 ΔFZD1-10 HEK293 T cells were seeded onto PDL-coated white 96-well cell culture plate with solid flat bottom (Greiner Bio-One). Next day, cells were transfected with 20 ng of SNAP-tagged receptor, 20 ng M50 Super 8× TOPFlash (Addgene, 12456), 2 ng pRL-TK Luc (Promega, E2241) and 58 ng pcDNA plasmid DNA to a final amount of 100 ng of plasmid DNA per well. Four hours after transfection, medium was changed to starvation medium (DMEM without FBS) containing either vehicle or 1000 ng/ml recombinant WNT-3A for FZD transfected cells or 100 nM SAG1.3 for SMO-transfected cells. Twenty-four hours after stimulation, cells were lysed gently shaking with 20 µl 1× Passive Lysis Buffer (Promega, E1910) for 15 min. Subsequently, 20 µl of LAR II (Promega, E1910) were added to all wells after which luminescence (580-80 nm) was read and then 20 µl of Stop & Glo (Promega, E1910) were added to all wells after which luminescence (480-80 nm) was read again with a CLARIOstar microplate reader (BMG). Data were analyzed using GraphPad Prism 6.
Lar 2
Manufactured by Promega
Sourced in Germany, United States
The LAR II is a laboratory equipment product designed for the measurement of light-producing reactions. It serves as a versatile luminescence detection system, providing consistent and accurate quantification of luminescent signals.
Automatically generated - may contain errors
Lab products found in correlation
Stop glo reagent, by Promega (7 mentions)
Passive lysis buffer (plb), by Promega (7 mentions)
Stop glo, by Promega (3 mentions)
Dual luciferase reporter assay system, by Promega (3 mentions)
Prl tk luc, by Promega (2 mentions)
M50 super 8 topflash, by Addgene (2 mentions)
96 well microplate, by Greiner (2 mentions)
Passive lysis buffer plb, by Promega (2 mentions)
Glomax multi detection system, by Promega (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Pdl coated white 96 well cell culture plate with solid flat bottom, by Greiner (1 mentions)
Dna lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Tcf reporter plasmid kit, by Merck Group (1 mentions)
Renilla luciferase assay, by Promega (1 mentions)
Prl cmv vector, by Promega (1 mentions)
Spectramax m3 microplate reader, by Molecular Devices (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Coelenterazine, by Nanolight Technology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
17 protocols using lar 2
1
Wnt Signaling Pathway Activation Assay
2
Dual Luciferase Assay for Wnt Signaling
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The dual luciferase assay was performed with the dual-luciferase®reporter assay system (Promega Corp., Madison, WI). All reagents were prepared as described by the manufacturer of the TCF Reporter Plasmid Kit (Millipore Corp., MA, USA). After the transfection complex was formed with FOP DNA, pRL-TK Vector renilla (Promega Corp., Madison, WI) and 50 μl of DNA Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA), the cells were seeded into 96-well plates with 100 μl/well. U2OS cells were divided into four groups: oleandrin (time): 50 nM oleandrin for 0, 24 and 48 h; oleandrin (concentration): 0, 25 and 50 nM oleandrin for 24 h, LiCl + oleandrin (time) and LiCl + oleandrin (concentration). For the LiCl groups, the cells were pretreated with 20 mM LiCl for 6 h to activate the Wnt signaling pathway. After the cells were lysed with PLB for 15 min, firefly luciferase reagent LARII (100 μl) and Stop & Glo Reagent (100 μl) (Promega Corp., Madison, WI) were added. The OD values of the TOP flash and the FOP flash were detected and the TOP/FOP ratio reflected the activity of the Wnt/β-catenin signaling pathway.
3
Luciferase Assay for Transfection
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Cells were harvested for luciferase activity 48 hours after transfection. Cells were lysed in 1X passive lysis buffer (Promega). To measure luciferase, LARII (Promega) was normalized by Renilla luciferase assay (Promega). All luciferase assays were performed in triplicate (n≥3).
4
Dual Luciferase Reporter Assay in HEK293 Cells
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HEK293 cells were transfected with a mixture of plasmids, encoding 1) for firefly luciferase, under the control of Thymidine Kinase minimal promoter and 2) firefly luciferase under the control of EF1 promoter ± the TRIB1 3’UTR. Twenty-four hours post-transfection, media was removed and cells were washed with PBS 1X twice and then lysed using 35 ml of 1X Passive Lysis Buffer (Promega); 5 ml of lysate was transferred onto a Nunc 384-well polystyrene white microplate. The substrates of Firefly luciferase (LAR II, Promega) and Renilla luciferase (Stop & Glo, Promega) were sequentially added to the cell lysates (1:1 ratio). From the same sample, luminescence was measured first at 560 nm for firefly luciferase and at 480 nm for Renilla luciferase using a microplate reader (Thermo Fisher Scientific). Each condition was plated in triplicate. All the readings were first normalized to the readings generated by non-transfected cells to subtract the luminescence background and then Renilla/Firefly ratio was calculated.
5
Dual-Luciferase Assay Protocol
6
Dual-Luciferase Assay Protocol
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At each time point, 50 μL of cell culture was removed, the cells were pelleted by centrifugation, and the supernatant was removed. Cells were resuspended in 300 μL 1X Passive Lysis Buffer (PLB, Promega). 20 μL of the resultant cell lysate was added to a 96-well microplate (Greiner Bio-One). The dual-LUC assay was performed by first adding 100 μL LAR II (Promega) to measure FLUC activity, then 100 μL of Stop & Glo reagent (Promega) was added to measure RLUC activity. The assay was performed and measurements were taken using a Promega Glomax Multi+ detection system.
7
Dual-Luciferase Reporter Assay in MEFs
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MEFs were seeded on 12-well plates. Cell density was 80% confluent on the day of transfection. MEFs were transfected using Lipofectamine LTX and PLUS (Invitrogen) according to the manufacturer’s instructions. 1 μg of indicated luciferase constructs were always co-transfected with 10 ng of the Renilla luciferase plasmid (pRL-CMVvector, Promega). After 24 hours, cells were lysed in 250μl Passive Lysis (Promega), 20μl lysate was added to 100μl LAR II (Promega) for luciferase activity, then 100μl Stop&Glo reagent (Promega) was added to the same well for measuring Renilla luciferase activity. Luciferase activities were measured in the spectraMax M3 microplate reader (Molecular Device, Sunnyvale, CA) using the dual-luciferase reporter assay system (Promega). Data were normalized for activity of Renilla luciferase. Three transfections of each construct plasmids were used in every assay.
8
Dual-Luciferase Reporter Assay Protocol
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Cells were co-transfected with firefly luciferase and Renilla luciferase plasmids for 48 h. Growth medium was then removed, and monolayers washed gently and thoroughly in PBS, to avoid disruption. PBS was complexly removed. Then 1×PLB (Promega) was added to each well, and plates rocked at room temperature for 15 min. 100 μl LAR II (Promega) was added to luminometer tubes, including 20 μl cell lysates. Absorbance was measured at 560 nm. 100 μl Stop & Glo reagents (Promega) were then added to each tube, and absorbance measured at 560 nm. Luciferase results were determined by dividing absorbance at 560 nm by absorbance at 470 nm.
9
Quantifying NQO1 Reporter Activity
10
Wnt Signaling Pathway Activation in HEK 293T Cells
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ΔFZD1–10 HEK 293T cells were
transfected in suspension (4 × 105 cells were transfected
in 1 mL) with 700 ng of HiBiT-tagged receptor,
250 ng M50 Super 8× TOPFlash (#12456; Addgene, Watertown, MA,
USA), and 50 ng pRL-TK Luc (#E2241, Promega, Fitchurg, WI, USA) and
seeded (50 μL) onto a poly(d -lysine)-coated white 96-well
cell culture plate with a solid flat bottom (Greiner BioOne). Next,
50 μL of complete DMEM medium was added to each well. Twenty-four
hours after transfection, the medium was changed to starvation medium
(DMEM without FBS) containing either 8.0 nM (300 ng/mL) WNT-3A or
vehicle, and 10 nM C59. Twenty-four hours after stimulation, cells
were lysed gently shaking with 20 μL 1× Passive Lysis Buffer
(#E1910; Promega, Fitchurg, WI, USA) for 15 min. Subsequently, 20
μL of LAR II (Promega, E1910) was added to all wells after which
luminescence (580–80 nm) was read, and then 20 μL of
Stop & Glo (Promega, E1910) was added to all wells after which
luminescence (480–80 nm) was read again with a CLARIOstar microplate
reader (BMG, Ortenberg, Germany).
transfected in suspension (4 × 105 cells were transfected
in 1 mL) with 700 ng of HiBiT-tagged receptor,
250 ng M50 Super 8× TOPFlash (#12456; Addgene, Watertown, MA,
USA), and 50 ng pRL-TK Luc (#E2241, Promega, Fitchurg, WI, USA) and
seeded (50 μL) onto a poly(
cell culture plate with a solid flat bottom (Greiner BioOne). Next,
50 μL of complete DMEM medium was added to each well. Twenty-four
hours after transfection, the medium was changed to starvation medium
(DMEM without FBS) containing either 8.0 nM (300 ng/mL) WNT-3A or
vehicle, and 10 nM C59. Twenty-four hours after stimulation, cells
were lysed gently shaking with 20 μL 1× Passive Lysis Buffer
(#E1910; Promega, Fitchurg, WI, USA) for 15 min. Subsequently, 20
μL of LAR II (Promega, E1910) was added to all wells after which
luminescence (580–80 nm) was read, and then 20 μL of
Stop & Glo (Promega, E1910) was added to all wells after which
luminescence (480–80 nm) was read again with a CLARIOstar microplate
reader (BMG, Ortenberg, Germany).
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