Plasmid containing ChREBP-luciferase was a kind gift from Dr Lawrence Chan, BCM. This was transfected using Lipofectamine 2000 (Life Technologies) into 22Rv1 KD and NT cells. Forty-eight hours later, luciferase signal was measured using a luminometer after adding Steady-Glo Luciferase reagent from Promega Inc. (cat # E2510) per the manufacturer's protocol (data were normalized using total protein content in the cells).
Steady glo luciferase reagent
Manufactured by Promega
Sourced in United States, United Kingdom
The Steady-Glo luciferase reagent is a product offered by Promega. It is a reagent designed for use in luciferase-based assays. The core function of the Steady-Glo luciferase reagent is to provide a stable and sustained luminescent signal for the detection and quantification of luciferase activity.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by GE Healthcare (2 mentions)
Glomax 96 luminometer, by Promega (2 mentions)
96 well black plates, by Avantor (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Glo lysis buffer, by Promega (1 mentions)
Dulbecco modified eagle medium (dmem), by GE Healthcare (1 mentions)
Resazurin sodium salt, by MP Biomedicals (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Protein a g column, by Thermo Fisher Scientific (1 mentions)
Protein l column, by Thermo Fisher Scientific (1 mentions)
Etoposide, by Enzo Life Sciences (1 mentions)
Penicillin streptomycin glutamine 100, by Thermo Fisher Scientific (1 mentions)
Hek293t 17 cell line, by American Type Culture Collection (1 mentions)
Bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Picogreen reagent, by Thermo Fisher Scientific (1 mentions)
Varioscan flash, by Thermo Fisher Scientific (1 mentions)
Centro lb 960, by Berthold Technologies (1 mentions)
20 protocols using steady glo luciferase reagent
1
Transient Transfection of ChREBP-Luciferase
2
Luciferase Assay for HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 2.5 × 105 HeLa cells were plated onto each well of the 6 MW plate the day before transfection. A total of 1 µg of pFLuc190UGA plasmid [19 (link),21 (link)] was transfected alone or co-transfected with 2 µg of FTSJ1 plasmid [17 (link)] by using Lipofectamine 2000 reagent (Invitrogen, Waltham, MA, USA), according to the provider’s instructions. A total of 1 µg of pFLuc-WT plasmid was transfected alone as a positive control. Twenty-four hours after transfection or co-transfection, HeLa cells were left untreated or treated for 24 h with 6 µm, 12 µm, or 24 µm for each NV. The next day, after two washes in DPBS 1X, cells were incubated for 5 min with 400 µL/well of the detection mix-Steady-Glo luciferase reagent (Promega, Madison, WI, USA). A total of 200 µL of cell suspension was then plated in duplicate onto a 96-well plate. Luciferase activity was then acquired with a luminometer.
3
Cell Culture Conditions for BJ Fibroblasts and HepG2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human foreskin fibroblast cell line (BJ fibroblasts, ATCC) was cultured in DMEM (high glucose) growth medium supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin. Human hepatocellular carcinoma cell line (HepG2, ATCC) was cultured in MEM growth medium (with Eagle’s basic salt solution) with the same supplements. All cells were cultured at 37 °C in a saturated humidity atmosphere with 5% CO2. DMEM (high glucose), MEM (with Eagle’s basic salt solution), penicillin-streptomycin 100× solution, fetal bovine serum (FBS) and trypsin 0.5% 10× solution were purchased from Hyclone, GE Healthcare Life Sciences. Alamar Blue stock solution was prepared by dissolving 1 g of resazurin sodium salt (MP Biomedicals) in 100 mL sterile PBS and filtered through a 0.22 µm filter. Stemfect mRNA transfection kit (Cat# 00–0069) was purchased from Stemgent. Steady-GLO luciferase reagent and GLO lysis buffer were purchased from Promega.
4
Pseudovirus Infection Assay in U87 Cells
5
PTC124 Effect on Luciferase Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cells were plated in a 12 multiwell plate, at a density of
1 × 105/mL in a volume of 1 mL per well, and transfected
with wild type (pFLuc) and mutant (pFLuc190UGA) plasmids
by using lipofectamine 2000 (Invitrogen). Cells were then incubated
for 24 h before the addition of 12 μM PTC124. After 24 h of
treatment, the cells were washed with PBS and incubated with the detection
mix Steady-Glo luciferase reagent (Promega). Aliquots of 200 μL
of cell suspension were plated in triplicate in a 96 well. Luciferase
activity was measured by a luminometer (Promega). Stably transfected
(H2B-GFP wt and H2B-GFP-opal) HeLa cells were selected by antibiotic
(blasticidin) resistance and characterized by PCR to verify the integrity
of the reporter gene. Expression of the fusion protein in live cells
after treatment with PTC124 (72 h) was monitored by fluorescence microscopy
(ZEISS). All of the examined cells underwent identical treatment with
the exception of drug concentration. Different brightness in microscopic
images has to be attributed to the confluence of the cells.
1 × 105/mL in a volume of 1 mL per well, and transfected
with wild type (pFLuc) and mutant (pFLuc190UGA) plasmids
by using lipofectamine 2000 (Invitrogen). Cells were then incubated
for 24 h before the addition of 12 μM PTC124. After 24 h of
treatment, the cells were washed with PBS and incubated with the detection
mix Steady-Glo luciferase reagent (Promega). Aliquots of 200 μL
of cell suspension were plated in triplicate in a 96 well. Luciferase
activity was measured by a luminometer (Promega). Stably transfected
(H2B-GFP wt and H2B-GFP-opal) HeLa cells were selected by antibiotic
(blasticidin) resistance and characterized by PCR to verify the integrity
of the reporter gene. Expression of the fusion protein in live cells
after treatment with PTC124 (72 h) was monitored by fluorescence microscopy
(ZEISS). All of the examined cells underwent identical treatment with
the exception of drug concentration. Different brightness in microscopic
images has to be attributed to the confluence of the cells.
6
T Cell Co-Stimulation and Checkpoint Inhibition Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Jurkat cell line engineered to express PD-1 and NFAT-Luciferase (Jurkat PD-1/NFAT/Luc) and Steady-Glo luciferase reagent were purchased from Promega (Madison, WI, USA). CHO cell line was engineered in-house to express T cell receptor activator (TCRA) and PD-L1 (CHO/PD-L1/TCRA) also referred to as APC. Raji cells endogenously expressing CD40 was engineered in-house to express NFkB/Luciferase (Raji/NFkB-Luc). All cell lines were routinely cultured in their respective cell culture medium. Assay ready cell (ARC) banks were generated and used in the experiments. All cell lines were tested for the absence of mycoplasma. APC culture medium contains DMEM (ThermoFisher Scientific, Waltham, MA, USA, Cat# 11995) supplemented with 10% heat inactivated (HI) FBS. Assay medium contains RPMI 1640 GlutaMAX, HEPES (ThermoFisher Scientific, Cat# 72400) supplemented with 10% HI FBS.
7
Evaluating NF-κB Pathway Modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
KG1a and U937 cell lines were transduced with lentivirus containing pGreenfire-NF-κB-responsive transcriptional elements-GFP/luciferase gene (Systems Bioscience) and selected with 2 μg/ml puromycin. Then the NFκB reporter cells were treated with pevonedistat or sapanisertib or both for timed exposures, lysed in passive lysis buffer (Promega) and analyzed for luciferase activity using Steady-GLO luciferase reagent (Promega) with a plate reader (Synergy 2, BioTek).
8
Budding Assay for Lassa Virus Z Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The luciferase-based budding assay was performed as described [40 (link)]. Briefly, 293T cells were seeded on poly-L-lysine-coated 12-well plates (3.5 × 105 cells/well). After overnight incubation, 2 µg of DNA of either pC-LASV-Z-Gaussia luciferase (GLuc) or pC-LASV-mutant Z[G2A]-GLuc were transfected using Lipofectamine 2000 (2.5 µL/µg of DNA). After 5 h, transfection mixtures were replaced with fresh media containing the indicated hit compounds. After 48 h, TCSs containing virion-like particles (VLPs) were harvested and clarified by low-speed centrifugation to remove cell debris. Aliquots (20 µL each) from TCS samples were added to 96-well black plates (VWR, West Chester, PA, USA), and 50 µL of SteadyGlo luciferase reagent (Promega) were added to each well. WCLs from the same samples were processed to determine cell-associated activity of GLuc. The luminescence signal was measured using the Berthold Centro LB 960 luminometer (Berthold Technologies, Oak Ridge, TN, USA). The activity (relative light units) of GLuc in TCSs and WCLs was used as a surrogate of Z expression. Budding efficiency was defined as the ratio ZVLP:(ZVLP+ZWCL).
9
Ruthenium-labeled Protein A/G/L Reagent
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Unlabeled protein A/G/L used for detection in the NHP TAb assay was purchased from BioVision (Milpitas, CA, USA). The ruthenium-labeled protein A/G/L reagent was prepared by reconstituting protein A/G/L according to manufacturer’s recommendation at 10 mg/ml in DPBS without calcium and magnesium. The concentration was adjusted to 1 mg ml−1 in DPBS and labeled with 10 nmol MSD Sulfo-NHS Ruthenium for 1 h at ambient temperature with rotation. The labeled protein A/G/L was purified by three cycles of filtration with Amicon-15 centrifugal filters using DPBS and stored at −80 °C in single-use aliquots. Tris Buffered Saline with 1% (w/v) Casein (TBS-C) was from BioRad (Hercules, CA, USA). The protein A/G columns, protein L columns, and bicinchoninic acid (BCA) protein assay kits were from Thermo Fisher Scientific (South San Francisco, CA, USA). Etoposide was from Enzo Life Sciences (Farmingdale, NY, USA), Steady-Glo Luciferase reagent was from Promega (Sunnyvale, CA, USA), fetal bovine serum (FBS) without heat inactivation was from HyClone (Logan, UT, USA), and Penicillin-Streptomycin-Glutamine (100 ×) was from Gibco (South San Francisco, CA, USA). The HEK293T/17 cell line was purchased from ATCC (Manassas, VA, USA). The GFP quantification kit was purchased from Cell Biolabs (San Diego, CA, USA).
10
Quantifying HIV-1 Infectivity via Luciferase Reporter
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!