The following reagents were purchased from Invitrogen (Carlsbad, CA): Dulbecco’s Modified Eagle Medium (DMEM) high glucose (cat# 11965–085), Roswell Park Memorial Institute (RPMI) 1640 (cat# 11875–085), DMEM high glucose without phenol red (cat# 31053–028), RPMI 1640 without phenol red (cat# 11835–030), Glutamax 200 mM (x100) (Cat# 35050), G418 (Geneticin® Selective Antibiotic, cat# 11811–031), and StemPro Accutase Cell Dissociation Reagent (cat# A11105–01). Tet System Approved FBS, US-Sourced (cat# 631101) and Doxycyline (cat# 631311) were purchased from Clontech (Mountain View, CA). Puromycin dihydrochloride from Streptomyces alboniger (cat# P8833–25MG) was purchased from Sigma-Aldrich (St. Louis, MO) and Steady-Glo substrate (cat# E2550) was purchased from Promega (Madison, WI).
Steady glo substrate
Manufactured by Promega
Sourced in United States
Steady-Glo substrate is a luminescent reagent used for the detection and quantification of various biomolecules, such as proteins, enzymes, and reporter genes, in cell-based assays. It provides a stable and sustained luminescent signal, enabling reliable and reproducible measurements.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco s modified eagle medium dmem high glucose, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 without phenol red, by Thermo Fisher Scientific (2 mentions)
Stempro accutase cell dissociation reagent, by Thermo Fisher Scientific (2 mentions)
Puromycin dihydrochloride from streptomyces alboniger, by Merck Group (2 mentions)
Dmem high glucose without phenol red, by Thermo Fisher Scientific (2 mentions)
Glutamax 200 mm x100, by Thermo Fisher Scientific (2 mentions)
Doxycyline, by Takara Bio (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Victor3 multilabel counter, by PerkinElmer (2 mentions)
Cell lysis reagent, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
Lipofectamine 2000, by Roche (1 mentions)
Modulus single tube luminometer, by Promega (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Steady glo luciferase assay system, by Promega (1 mentions)
Topcount, by PerkinElmer (1 mentions)
Victor3, by PerkinElmer (1 mentions)
8 protocols using steady glo substrate
1
Cell Culture Protocol with Selective Reagents
2
Cell Culture Protocol with Selective Reagents
3
Adenovirus Neutralization Antibody Assay
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Adenovirus-specific neutralization antibody (NAb) titers using mouse serum samples were conducted as previously described (31 (link)). Briefly, serum was 2-fold serially diluted in a 96-well flat-bottom plate, with the exception of the last column, which served as the maximum-infection control. Replication-incompetent recombinant Ad-Luc reporter construct viruses were added to the plate, followed by the addition of A459 cells. The plates were incubated for 24 h at 37°C in 10% CO2. After incubation, the medium was removed and 100 μl of phosphate-buffered saline (PBS) and 100 μl of Steady-Glo substrate (Promega) were added to the wells. The luciferase (Luc) activity in the cells was measured with a Victor 3 multilabel counter (PerkinElmer, Waltham, MA). Neutralization titers were defined as the maximum serial dilution where 90% of the virus was neutralized by the serum.
4
Transfection and Silencing Assays in Cell Lines
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HEK293T cells were co-transfected with reporter vectors along with other expression vectors or molecular clones using Lipofectamine 2000 (Invitrogen, USA) and harvested 36 h post-transfection for luciferase assay. The cells were lysed in cell lysis reagent (Promega, USA) and luciferase assays were performed using Steady-Glo substrate (Promega, USA) as described earlier (30 (link)). Jurkat cells were transfected using x-treme gene HP DNA transfection reagent (Roche Applied Bioscience, Germany).
For silencing studies, cells were first transfected with siRNA using Lipofectamine 2000 as per the manufacturer's instructions, followed by second transfection (reporter plasmid/ expression vectors) or infection as described earlier (52 (link)). Cells were harvested 48 h post-transfection/infection. Knockdown was confirmed by immunoblotting with respective antibodies. Immunoblotting with GAPDH served as the loading control.
For silencing studies, cells were first transfected with siRNA using Lipofectamine 2000 as per the manufacturer's instructions, followed by second transfection (reporter plasmid/ expression vectors) or infection as described earlier (52 (link)). Cells were harvested 48 h post-transfection/infection. Knockdown was confirmed by immunoblotting with respective antibodies. Immunoblotting with GAPDH served as the loading control.
5
AvBD9 Promoter-Driven Luciferase Assay
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HTC cells were seeded overnight in 24-well tissue culture plates before being transfected with 50 ng/well of different AvBD9 promoter-driven luciferase reporter plasmids using FuGENE HD Transfection Reagent (Promega). After 24 h, cells were stimulated in duplicate with or without 8 mM sodium butyrate for another 24 h. Luciferase activity was measured by adding an equal volume of Steady-Glo Substrate to each well for 10 min using Steady-Glo Luciferase Assay System (Promega) according to the manufacturers' instructions. The luminescence was detected using Modulus Single-Tube Luminometer (Turner Biosystems, Sunnuvale, CA).
6
Ad5-Specific Neutralizing Antibody Assay
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Ad5-specific neutralizing antibodies were measured using a luciferase-based neutralization assay as previously described (51 (link)). Briefly, serum from treated mice was serially diluted, Ad5.Luc virus was added, and the mixture was added to A549 cells. Following 24 h of incubation, medium was removed and 100 μl PBS and 100 μl Steady-Glo substrate (Promega) were added to wells. Luminescence was read on a Victor 3 multilabel counter (PerkinElmer), and the 90% inhibitory concentration (IC90) was determined (dilution of serum at which 90% of Ad5.Luc was neutralized).
7
Screening and Characterization of BNBC
8
Luminescence-based Nrf2 Pathway Activation
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100 µL of a cell suspension (10 5 cells/mL) were added to 96 well plates and incubated for 24 ± 4 h at 37 °C in a humidified atmosphere with 5% CO 2 . Afterwards, the medium got replaced by 50 µL of the sample or dissolved chemicals. The plates were covered with sealing film and incubated for 6 h at 37 °C and 5% CO 2 .
For measurement of the viability, Steady-Glo ® substrate (Promega) was dissolved in the corresponding buffer and further diluted 1:10 with RO-H 2 O to a final concentration of 1.54 mg/mL. 50 µL of the Steady-Glo ® solution were added to the wells and luminescence measured with a plate reader (Victor 3 , Perkin Elmer).
Afterwards, for measurement of sensitization, the Nano-Glo ® Luciferase substrate was diluted 1:50 in the corresponding buffer and further diluted 1:10 in PBS containing 1.5% EDTA. 50 µL of this solution was added to the wells and luminescence measured with a plate reader (Victor 3 , Perkin Elmer).
Fold inductions were calculated as the ratio of the relative light units of the samples to the relative light units of untreated PBS + 1% DMSO. A fold induction above 2 was counted as significant activation of the ARE-Nrf2 pathway.
For measurement of the viability, Steady-Glo ® substrate (Promega) was dissolved in the corresponding buffer and further diluted 1:10 with RO-H 2 O to a final concentration of 1.54 mg/mL. 50 µL of the Steady-Glo ® solution were added to the wells and luminescence measured with a plate reader (Victor 3 , Perkin Elmer).
Afterwards, for measurement of sensitization, the Nano-Glo ® Luciferase substrate was diluted 1:50 in the corresponding buffer and further diluted 1:10 in PBS containing 1.5% EDTA. 50 µL of this solution was added to the wells and luminescence measured with a plate reader (Victor 3 , Perkin Elmer).
Fold inductions were calculated as the ratio of the relative light units of the samples to the relative light units of untreated PBS + 1% DMSO. A fold induction above 2 was counted as significant activation of the ARE-Nrf2 pathway.
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