The human cyclin A promoter (containing 1 GC box and 1 TATAAT box) was cloned into the pGL3 basic firefly luciferase vector (Thermo Scientific) and then transfected into PFDN1-overexpressed cells. The specific primers for promoter sequences are listed in supplementary Table S5 . Cell lysates were prepared 24 h after transfection in reporter lysis buffer (Promega, Madison, WI, USA) and luciferase substrate was added. The luciferase activity was recorded on a luminometer (Veritas, Promega).
Veritas
Manufactured by Promega
Sourced in United States
Veritas is a high-performance luminometer designed for sensitive and accurate measurement of luminescent signals. It is capable of detecting a wide range of luminescent assays, including luciferase-based reporter gene assays, ATP-based cell viability assays, and protein-protein interaction assays. The Veritas luminometer provides precise and reliable data, making it a valuable tool for researchers in various life science applications.
Automatically generated - may contain errors
Lab products found in correlation
White 96 well plate, by SPL Life Sciences (3 mentions)
Pgl3 basic firefly luciferase vector, by Thermo Fisher Scientific (2 mentions)
Reporter lysis buffer, by Promega (2 mentions)
Fugene 6 reagent, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Ldh glo, by Promega (1 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Amaxa mouse macrophage nucleofector kit, by Lonza (1 mentions)
Luciferase assay reagent, by Promega (1 mentions)
Lysis buffer, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Celltiter glo 2.0 viability assay, by Promega (1 mentions)
Prism 8, by GraphPad (1 mentions)
Complete edta free, by Roche (1 mentions)
Coelenterazine h, by Fujifilm (1 mentions)
Ms 8096w, by Sumitomo Bakelite (1 mentions)
L 15 medium without phenol red, by Thermo Fisher Scientific (1 mentions)
Pei max, by Polysciences (1 mentions)
22 protocols using veritas
1
Human Cyclin A Promoter Luciferase Assay
2
Homo Cdh1 Promoter Luciferase Assay
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The Homo Cdh1 wt, mut1, mut2, and mut1-2 promoter sequences were cloned into the pGL3 basic firefly luciferase vector (Thermo Scientific) and subsequently transfected into 293T cells. Following a 24-h incubation period, cell lysates were prepared in reporter lysis buffer (Promega, Madison, WI, USA) and luciferase substrate was introduced. The resulting luciferase activity was measured using a luminometer (Veritas, Promega).
3
Measuring TGF-β1 Transcriptional Activity
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NMuMG cells were transfected with the firefly luciferase (Luc) reporter FN1-Lux (1 μg) and pCMV–Renilla reniformis luciferase (0.025 μg) (Rl; Promega) in 6-well plates (106 cells/well) using FuGENE 6 reagent and according to the manufacturer’s protocol. The next day, cells were transferred into a 48-well plate (2 × 104 cells/well). The cells were treated with 2 ng/ml TGF-β1 for 24 h. Luc and Rl activities were determined using the Dual-Luciferase reporter assay system (Promega), according to the manufacturer’s protocol, in a microplate luminometer (Veritas; Promega). Firefly activity was normalized to Renilla activity and presented as relative luciferase units. All assays were performed in triplicates, and each experiment was repeated at least twice.
4
Transepithelial Electrical Resistance and Cell Viability
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TEER was measured with a volt-ohmmeter and electrode probe (EVOM and STX2; World Precision Instruments, Sarasota, FL, USA). Details on obtaining reproducible resistance readings are available at: https://medicine.umich.edu/sites/default/files/content/downloads/Measuring_Transepithelial_Electrical_Resistance.pdf .
Cell death was measured on cultures at least several weeks after last OS feeding using a cytotoxicity assay (LDH-Glo, #J2380; Promega, Madison, WI, USA) according to manufacturer's instructions. Supernatant (24-hour incubation) was diluted 1:100, and 50 μL of the dilution was measured on a luminometer (Veritas; Promega), subtracting all values from the signal derived from fresh media.
Cell death was measured on cultures at least several weeks after last OS feeding using a cytotoxicity assay (LDH-Glo, #J2380; Promega, Madison, WI, USA) according to manufacturer's instructions. Supernatant (24-hour incubation) was diluted 1:100, and 50 μL of the dilution was measured on a luminometer (Veritas; Promega), subtracting all values from the signal derived from fresh media.
5
Transient Transfection Assay for Reporter Gene Expression
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Cells were transfected with either IL-10-luciferase reporter construct (Adgene) or PPRE-luciferase reporter construct (Qiagen, USA, Cat # CCS-3026L), using Amaxa Mouse Macrophage Nucleofector Kit from Lonza (Cat # VPA-1009) following manufacturer's instructions. Reporter assay was carried out 24 h post-transfection using dual luciferase assay kit (Promega, Cat # E1910) following manufacturer's protocol. Briefly, cells were washed with PBS followed by lysis with 1X passive lysis buffer. The lysate was cleared by brief centrifugation and firefly luciferase reporter activity in the clear supernatant was measured using a luminometer (Veritas, Promega). Renilla luciferase activity in the same lysate was used to assess transfection efficiency.
6
Luciferase Assay of PGC-1α Promoter
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The SH-SY5Y cells were transfected with 0.8 µg/well of PGC-1α-delta-CRE-promoter-luciferase reporter plasmid (the CREB-binding sequences were deleted), PGC-1α-promoter-luciferase plasmid (encoding wild type PGC-1α promoter sequences upstream of luciferase reporter gene), PGL negative control plasmid or CMV-luciferase positive control plasmid respectively using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA). Cells transfected with the plasmid DNA mixtures were cultured for 48 h. After washing with PBS, the cells were lysed with the lysis buffer (Promega, USA). The cell lysates were mixed with Luciferase Assay Reagent (Promega, USA) in 96-well plate, and the light was measured using a 96-well microplate luminometer (Veritas, Promega, USA) [30] (link).
7
Cell Proliferation Assay with KD Genes
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Plate reader–based proliferation assays were performed on cells previously transduced with PLAT, PLAU, or KLK6 KD virus. The cells were under selection for 5 days after virus addition prior to setting up the experiment. The day before the assay, 5000 cells/well in 100 μl complete media were plated into 96 well plates and incubated for 18 h. For diminazene, gabexate mesylate, and hydroxystilbamidine treatment experiments, cells were seeded at 5000 cells/well and incubated with vehicle only (DMSO at final concentration of 1%) or with each drug at the indicated final concentrations for 18 h in complete media before commencement of the assay. The assay was carried out following the manufacture’s protocol (Promega CellTiter Glo 2.0 viability assay). Cells were lysed and incubated with Promega reagent and luminescent signal was acquired following the provided standard Promega protocol (Veritas, Turner BioSystems). Data were analyzed using GraphPad Prism 8.0.
8
Dengue Virus RNA Polymerase Assay
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Example 166
Dengue RNA-Dependent RNA Polymerase Reaction Conditions
RNA polymerase assay was performed at 30° C. using 100 μl reaction mix in 1.5 ml tube. Final reaction conditions were 50 mM Hepes (pH 7.0), 2 mM DTT, 1 mM MnCl2, 10 mM KCl, 100 nM UTR-Poly A (self-annealing primer), 10 M UTP, 26 nM RdRp enzyme. The reaction mix with different compounds (inhibitors) was incubated at 30° C. for 1 hour. To assess amount of pyrophosphate generated during polymerase reaction, 30 μl of polymerase reaction mix was mixed with a luciferase coupled-enzyme reaction mix (70 μl). Final reaction conditions of luciferase reaction were 5 mM MgCl2, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 200 μU ATP sulfurylase, 5 M APS, 10 nM Luciferase, 100 M D-luciferin. White plates containing the reaction samples (100 μl) were immediately transferred to the luminometer Veritas (Turner Biosystems, CA) for detection of the light signal.
9
RNA Polymerase Inhibitor Screening Assay
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Example 26
RNA polymerase assay was performed at 30° C. using 100 μL reaction mix in 1.5 mL tube. Final reaction conditions were 50 mM Hepes (pH 7.0), 2 mM DTT, 1 mM MnCl2, 10 mM KCl, 100 nM UTR-Poly A (self-annealing primer), 10 μM UTP, 26 nM RdRp enzyme. The reaction mix with different compounds (inhibitors) was incubated at 30° C. for 1 hr. To assess amount of pyrophosphate generated during polymerase reaction, 30 μL of polymerase reaction mix was mixed with a luciferase coupled-enzyme reaction mix (70 μL). Final reaction conditions of luciferase reaction were 5 mM MgCl2, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 200 μU ATP sulfurylase, 5 μM APS, 10 nM Luciferase, 100 μM D-luciferin. White plates containing the reaction samples (100 μL) were immediately transferred to the luminometer Veritas (Turner Biosystems, CA) for detection of the light signal.
10
Dengue Virus RNA Polymerase Assay
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Example 50
Dengue RNA-Dependent RNA Polymerase Reaction Conditions
RNA polymerase assay was performed at 30° C.; using 100 μl reaction mix in 1.5 ml tube. Final reaction conditions were 50 mM Hepes (pH 7.0), 2 mM DTT, 1 mM MnCl2, 10 mM KCl, 100 nM UTR-Poly A (self-annealing primer), 10 μM UTP, 26 nM RdRp enzyme. The reaction mix with different compounds (inhibitors) was incubated at 30° C. for 1 hour. To assess amount of pyrophosphate generated during polymerase reaction, 30° C. of polymerase reaction mix was mixed with a luciferase coupled-enzyme reaction mix (70 μl). Final reaction conditions of luciferase reaction were 5 mM MgCl2, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 200 μU ATP sulfurylase, 5 μM APS, 10 nM Luciferase, 100 μM D-luciferin. White plates containing the reaction samples (100 μl) were immediately transferred to the luminometer Veritas (Turner Biosystems, CA) for detection of the light signal.
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