A luciferase adenoviral expression vector (AdNFkB-Luc) (Vector Biolabs) controlled by a promoter containing five repeats of the NFkB enhancer element (TGGGGACTTTCCGC) was used to infect HCAECs that were simultaneously infected with a β -galactosidase expression vector (Vector Biolabs). HCAEC were infected with adenovirus for 24 h prior to treatments. Cell lysates were obtained using the reporter lysis buffer (Promega) and promptly used for the luciferase and β-galactosidase enzyme assays (Promega). Firefly luciferase activity was measured using the 20/20n luminometer (Turner Biosystems), while β-galactosidase activity was measured by absorbance detection at 420 nm (SPECTRA MAX 190, Molecular Devices).
β galactosidase enzyme assay
Manufactured by Promega
Sourced in United Kingdom
The β-galactosidase enzyme assay is a colorimetric assay used to detect and quantify the presence of the β-galactosidase enzyme. The assay utilizes a chromogenic substrate that is cleaved by β-galactosidase, resulting in a color change that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Reporter lysis buffer, by Promega (4 mentions)
Psv β galactosidase control vector, by Promega (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Luciferase activity assay, by Promega (3 mentions)
Luciferase assay, by Promega (3 mentions)
Luciferase assay kit, by Promega (2 mentions)
Lipofectamine plus, by Thermo Fisher Scientific (2 mentions)
Luciferase assay reagent, by Promega (2 mentions)
Pnfκb luc vector, by Takara Bio (2 mentions)
Psv β galactosidase β gal control vector, by Promega (2 mentions)
N luminometer, by Promega (1 mentions)
Ad nfkb luc, by Vector Biolabs (1 mentions)
Spectramax 190, by Molecular Devices (1 mentions)
Luciferase, by Promega (1 mentions)
Synergy h1 microplate reader, by Synergy Software (1 mentions)
Smartspec 3000 spectrophotometer, by Bio-Rad (1 mentions)
Lipofectamine, by Promega (1 mentions)
Td 20 20, by Turner Designs (1 mentions)
Psv β gal, by Promega (1 mentions)
Luciferase assay system, by Promega (1 mentions)
15 protocols using β galactosidase enzyme assay
1
NFkB-Mediated Gene Expression Quantification
2
Transcriptional Activity of MEF2D Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MEF2D variants with modified dynamical properties were cloned into pCMV-HA eukaryotic expression vector (Genscript, Piscataway, NJ, USA). To determine the transcriptional activity of Mef2D variants, MCK-Luc reporter plasmid (Addgene, Watertone, MA, USA) and pSV-β-galactosidase control vector (Promega) were used together with the variant plasmids. Control and MEF2D-KO cells (description for the generation of KO cultures is in the section above) were transfected with the vectors of the variants using Lipofectamine 2000 transfection reagent (Life Technologies, Carlsbad, CA, USA). 48 hours later Luciferase and galactosidase activity were determined by Luciferase and β-galactosidase enzyme assays (both from Promega) following the manufacturer´s instructions. Luminescent and absorbance data were collected by Synergy H1 microplate reader to quantify the transcriptional activity of the samples. Microsoft Excel 2016 was used to process transcription and differentiation data.
3
NF-κB Activation in VSMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Activation of nuclear factor-κB (NF-κB) was determined in the VSMCs after transfection with a reporter plasmid containing the luciferase reporter gene linked to five repeats of the NF-κB binding sites. The VSMCs (1 × 105 cells/well) were plated in 24-well plates and grown to about 70% confluence. Cells were then transiently cotransfected with 1 µg of NF-κB–luciferase reporter plasmid and 1 µg of β-galactosidase plasmid using Lipofectamine plus (Invitrogen, Carlsbad, CA, USA). At 6 hours post-transfection, cells were starved for 48 hours before stimulation with lysoPC. Transfected cells were exposed to 5 µM lysoPC for the indicated time periods. Luciferase activity was measured using a luciferase assay kit (Promega, Madison, WI, USA) with signal detection for 5 seconds in a luminometer (Panomics Inc., Fremont, CA, USA). A β-galactosidase enzyme assay (Promega) was used to determine the β-galactosidase activity with a SmartSpec 3000 spectrophotometer at 420 nm. The results are expressed relative to the NF-κB activity compared with controls after normalizing for β-galactosidase activity and protein concentration.
4
NF-κB Activation in VSMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To examine the nuclear factor (NF)-κB activation, the VSMCs were transfected with a reporter plasmid containing the luciferase reporter gene linked to five repeats of the NF-κB binding sites, as previously described [15 (link)]. Briefly, the VSMCs (1 × 105 cells/well) were cultured to approximately 70% confluence in 24-well plates. Cells were then transiently co-transfected with 1 µg of NF-κB-luciferase reporter plasmid and 1 µg of β-galactosidase plasmid using Lipofectamine plus (Invitrogen, Carlsbad, CA, USA). At 6 hours after transfection, cells were starved for 48 hours, and then exposed to 15 µM lysoPC for the indicated time periods. Luciferase activity was measured with a luciferase assay kit (Promega, Madison, WI, USA) with signal detection for 5 seconds in a luminometer (Panomics Inc., Fremont, CA, USA). A β-galactosidase enzyme assay (Promega) was applied to determine the β-galactosidase activity at 420 nm with a SmartSpec 3000 spectrophotometer (Bio-Rad). The results are expressed relative to the NF-κB activity compared with controls after normalizing for β-galactosidase activity and protein concentration.
5
Transient Transfection Luciferase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transient transfection of luciferase reporter plasmids was performed using lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. Cells were plated in 12-well tissue culture plates at 2 × 105 per well for overnight. Nucleic acid-lipofectamine 2000 mixtures containing 0.5 μg of luciferase reporter vectors, 0.5 μg of pSV-β-galactosidase control vector (Promega) and 0.5 μg of expressing vectors or 40 nM siRNA were exposed to cells. After 24 h, cell lysates were harvested and assayed for the Luciferase activity assay (Promega), performed according to the protocol recommended by the supplier. To normalize the transfection efficiency, the same cell lysates were also assayed for β-galactosidase activity using the β-Galactosidase enzyme assay (Promega).
6
Measuring β-GAL Activity in Tissue Homogenates
7
NF-κB Activation Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The NF-κB-luciferase reporter system that was used to quantify the NF-κB activation following TLR stimulation was described previously (20 (link)). In brief, cells were washed twice with Dulbecco’s PBS and 100 μl of Reporter Lysis Buffer (Promega, Leiden, The Netherlands) was added to each well after which the plate was stored o/n at −80oC. Luciferase activity was measured by mixing 50 μl of Luciferase Assay Reagent (Promega) to 20 μl of defrosted cell lysate followed by direct measurement in a luminometer (TD-20/20, Turner Designs, Sunnyvale, CA, USA). The β-galactosidase enzyme assay (Promega) was performed by adding 50 μl of cell lysate to 50 μl of 2× β-galactosidase assay buffer as indicated by the manufacturer’s instructions. Luciferase values were normalized against the β-galactosidase values to obtain relative luciferase units (RLU). Results are expressed as percentage of maximal response of TLR-transfected cells with its cognate ligand. In experiments that included TNFα, TNFα addition was considered the maximum response.
8
Regulation of FOXC1 Promoter Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDA-MB-468 cells were transfected with the pGL4-FOXC1 promoter reporter construct and the β-galactosidase expression vector pSV-β-Gal (Promega Madison, WI) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the instruction manual. β-Galactosidase enzyme activity was detected using the β-Galactosidase enzyme assay system with reporter lysis buffer (Promega, Madison, WI). For co-transfection, 500 ng of Flag-ERK2, HA-Myr-Akt1, or HA-Myr-Akt3 constructs were added along with 100 ng of the 2-kb human FOXC1 promoter reporter construct pGL4-FOXC1. For the small interfering RNA (siRNA) experiment, MDA-MB-468 cells were transfected with 30 nM human FOXC1 and p65 siRNA for 48 h, and then treated with EGF for 24 h.
9
Modulation of IL-6 and COX-2 Transcription
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IEC-6 cells were seeded at a density of 1 × 105 cells per well in 24-well plates and grown to 80% confluence in complete growth medium. Cells were transiently cotransfected with 0.8 μg/well of translucent IL-6 or Cox-2 reporter vector. IL-6-luciferase reporter construct were gift from Dr. Seong Ho Jeon (Hanllym University, Chooncheon, Korea) and Cox-2-luciferase reporter construct were gift from Dr. Byung Ju Lee (Ulsan University Hospital, Ulsan, Korea). After 24 hours of transfection, cells were pretreated with cerulenin or cyclopamine for 1 hour and followed by stimulating with TNF-α for 6 hours. The cell were then washed with PBS and lysed in 1x reporter lysis buffer (Promega). The lysed cell extract (20 μl) was mixed with 100 μl of luciferase assay reagent (Promega) and the luciferase activity was measured by the luminometer (MDS Analysis Tecnologies). The β-galactosidase assay was done according to the supplier's instructions (Promega β-galactosidase enzyme assay) for normalizing the luciferase activity. All data are expressed as the percentage of activity relative to basal activity.
10
Enzymatic Analysis of β-Galactosidase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The β-Galactosidase enzyme assay was purchased from Promega (Southampton, UK) and was performed according to the manufacturer’s instructions.
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!