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NF-κB Reporter Lentivirus
$595.00
NFκB Reporter Lentivirus (Pan): High-quality lentiviral reporter system that provides a sensitive fluorescent (GFP/RFP) or luminescent (Luc) readout for human/mouse nuclear factor kappa B (NF-kB, NF-kappa B) activity in mammalian cells. The construct contains a minimal TATA promoter and tandem repeats of NFκB DNA-binding elements that drive expression of the reporter when bound to NFκB. An optimized mini-enhancer amplifies NFκB-dependent reporter expression resulting in a strong fluorescent/luminescent signal that can be used to readout NFκB pathway activity. An EF1a-driven selection marker (Puro/BSD) allows the user to easily create stable cell lines. This NFkB reporter is supplied as high-titer lentiviral particles which are VSV-G pseudotyped and ready-to-transduce a broad range of cell types. The NFkB Reporter lentiviral particles are purified by PEG precipitation and sucrose gradient centrifugation, serum-free, and ideal for studying NFκB signaling pathway activity in difficult-to-transfect cells including primary and/or thawed cells.
Our PhD-level staff is ready to assist you. Email us to discuss your project or obtain help selecting the right product for your research.
Email: info@lipexogen.com
Available Options:
Specifications
Key Advantages
- High Sensitivity – LipExoGen’s NFκB Reporter lentiviral particles are made using a novel vector platform based on the third generation system. Tandem repeats of the transcription factor’s response elements are arranged upstream of a minimal TATA promoter-driven reporter, and downstream of an optimized minimal enhancer of the human CMV promoter. When the signal pathway/TF is activated, the mini enhancer synergizes with TF binding to the response elements (up to 8 repeats in some products, depending on strength of reporter activation) to amplify expression of the fluorescent (GFP/RFP) or luciferase (Luc) reporter, with minimal enhancement of background. As a result, the reporter system provides a highly sensitive readout for signaling pathway or specific transcription factor activation in human and mouse cells.
- Easily Establish Stable Reporter Cell Lines – The reporter lentiviral particles are ultra-purified and concentrated to high-titer by PEG purification and sucrose gradient centrifugation to allow for efficient transduction of difficult-to-transfect cells, including primary and/or freeze-thawed cells. Stable cell lines are easily generated through puromycin or blasticidin selection.
- Discovery Made Easy – Signal pathway or specific transcription factor activity can be detected by fluorescence, making LipExoGen TF Reporter lentiviral particles more practical than traditional luciferase reporters and/or biochemical assays. Pathway/TF activation can be readout directly by fluorescence microscopy in living cell cultures, thus paving the way for unexpected discoveries.
- Readout On Flow – Fluorescent reporter activation can also be readout by flow cytometry, providing more versatility in data acquisition for labs with different instruments.
- Best Value – LipExoGen lentiviral particle products are made using optimized lentiviral vectors developed in-house, which allows us to provide the highest quality products while retaining competitive prices. These high-titer lentiviral particles feature a highly sensitive fluorescent reporter system which has been validated to read out the indicated transcription factor activity.
- Completely Customizable – You can easily request a modified version of this product, such as a dual-reporter or a different reporter not listed here, by contacting us.
Details:
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| LTV-0002 | |
| High-titer Lentiviral Particles, third generation, VSV-G pseudotyped | |
| Serum-free RPMI-1640, frozen solution | |
| PEG precipitation and sucrose gradient centrifugation | |
| One vial | |
| 380 μl/vial | |
| 1-5×10^6 TU/vial, depending on the variant | |
| 3×10^8 VP/ml | |
| -80oC | |
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| NF-κB signaling pathway | |
| Species | Human/mouse |
| Tandem repeats of response elements coupled to minimal TATA promoter and upstream proprietary enhancer | |
| NF-κB response element | |
| GFP, RFP, or Firefly Luciferase | |
| Promoter (Driving Selection Marker) | EF1α |
| Puromycin (Puro) or Blasticidin (BSD), or a fluorescent protein for select products | |
| Monitor NFκB pathway activation. Screen for activators or inhibitors of the NFκB pathway with fluorescence or luciferase. The fluorescent reporters enable convenient readout using flow cytometry, fluorescence microscopy, etc. |
*Based on infectivity on HEK293FT cells transduction units (TU).
Product Data
Figure 1 (thumbnail). HEK293FT cells were transfected with NFkB-TAG-Puro for 24 hrs, then stimulated with TNFα or PBS for 12 hrs. GFP reporter activation was then visualized by fluorescence microscopy.
Figure 2. NFκB-TAG-Puro Reporter Expression in RAW264.7 Stable Cell Line RAW264.7 mouse macrophage cells were thawed and briefly expanded in culture prior to spin transduction with NFκB-TAG-Puro lentivirus (LTV-0002-1). A stable cell line was established using puromycin selection. The images show activation of the GFP reporter in response to 8 h stimulation with with LPS (10 ng/ml) or PBS.
Figure 3. Comparison of NFκB reporter (pan) and NFκB (p65/p65) reporter activity in response to TNFα and REL-A (p65) overexpression. HEK293FT cells were transfected (24-36 h) with the pan NFkB reporter (LTV-0002, top panels) or NFκB p65/p65 reporter (LTV-0070, bottom panels). At the same time, some of the cells were co-transfected with empty vector (pcDNA, left) or an expression vector for human REL-A (p65, middle). After the transfection period of 24-36 h, cells on the right were stimulated with TNFα for 8 h, and then fluorescent images were acquired for all of the cells. Top panel shows the general activation of NFκB reporter by either TNFα or overexpression of p65, which is comparable. Bottom panels show the preferential activation of the p65 reporter activity in absence of TNFα and p65 overexpression, and minor activation by TNFα alone. This product is supplied as pre-packaged lentiviral particles with your choice of reporter and selection marker. REL-A (p65) cDNA expression vector is also available upon request. For more information, please email us.
Figure 4. NFkB-TAG-Puro THP1 stable cell line response to LPS treatment. THP1 cells were transduced with NFκB-TAG-Puro lentivirus (LTV-0002-1S) and selected with puromycin to establish a stable cell line. Fluorescence microscopy (A) and flow cytometry (B) were done after 48 h of treatment with LPS (right panels in A and blue shaded line in B) or PBS as control (left panels in A, dashed line in B). Bright field images are include in A to show the entire cell density in the fluorescent frame (the NFkB reporter was activated in appx 75% of the cells under these conditions).
Figure 5. NFκB-TAR-GFP HEK293FT cells were transfected for 16 h with NFκB-TAR-GFP, which expresses RFP under control of NFκB response elements and constitutively expressed GFP driven by EF1a. The Subsequently, 50 ng/ml of human TNFα was added for an additional 16 hours. Afterward, pictures were taken. In this construct, GFP is expressed constitutively whereas RFP is induced by the response elements. It could be useful for experiments where FACS sorting is desired instead of drug selection. For example, some cells which are too sensitive to prolonged culture after viral infection. In this case, the GFP allows the user to control for the transduction efficiency which will vary depending on your cell line and experimental conditions.
Figure 6. NFκB-TAB-Puro HEK293FT cells were transfected with NFκB-TAB2-Puro for 24 hours before being treated with 10 ng/ml TNFα for an additional 16 hours. This reporter expresses BFP2 as the reporter and puromycin as the selection marker.
Figure 7. HEK293FT cells were transfected with NFκB-RLuc-Puro, which expresses Renilla luciferase under control of NFκB response elements, and puromycin as the selection marker. The cells were lysed and lysate was measured using the DLR Assay from Promega (Cat. E1910). 10 ul of lysate + 50 ul of substrate mixture (each). Luminescence was recorded on a Spectramax iD3 with 1 s integration time using appropriate wavelengths for the FLuc and RLuc parts of the assay respectively.
Have questions about this product? Need a custom modification or stable cell line? Email us and we will respond the same day.
Email: info@lipexogen.com
Background
The NF-κB pathway is a crucial cellular signaling system that regulates immune and inflammatory responses, cell proliferation, and survival. Its activation leads to the transcription of genes involved in immune defense, cell growth, and anti-apoptotic processes. Dysregulation of this pathway is linked to various diseases, including cancer, autoimmune disorders, and chronic inflammation.
LipExoGen’s NFkB Reporter Lentivirus provides researchers with a powerful tool to study and manipulate the NFκB signaling pathway in cellular models. By incorporating a reporter system that fluoresces or luminesces in response to NFκB activation, it allows for real-time observation and quantitative analysis of pathway activation. This makes it an invaluable resource for developing therapeutic strategies targeting NF-κB-related pathways, ensuring researchers can monitor the effects of genetic or pharmacological manipulations on NF-κB activity in diverse cell types.
Vector Diagram
Related Products
NFkB Reporters
NFκB Reporter Lentivirus (specific for p65 homodimer)
NFκB (p52:RelB) Reporter Lentivirus
NFκB Pathway Inhibitors
Recommended Controls
Reporter Negative Control Lentivirus – Ready-to-transduce lentiviral particles expressing minimal TATA box-driven reporter. The construct is the same as the TF Reporters except that it lacks the transcriptional response elements which drive signal pathway/TF-specific reporter expression. The reporter negative control lentiviral particles allow to establish a baseline for background reporter activity and determine specificity of any treatments to activate the reporter.
Reporter Positive Control Lentivirus – Ready-to-transduce lentiviral particles with constitutive expression of the reporter. The reporter positive control lentivirus is useful for transduction optimization studies, especially if the cells are very sensitive or difficult-to-transduce.
Renilla Luciferase Internal Control Lentivirus – Ready-to-transduce lentiviral particles expressing minimal TATA box-driven Renilla luciferase (RLuc).
Publications
Custom Orders
If you require a modification to one of our products, such as a change in reporter or other vector component, please contact us. Examples of customization options are shown in the table below. Feel free to request something not in the table.
Additional Custom Service Options
- Send us your cells and we can establish a stable NFkB reporter cell line for you using this product. Learn more.
- ORF cDNA plasmids featured in the product figures are available upon request.
- Ultra-high concentration virus can be provided upon request in your choice of medium and volume (i.e. for in vivo applications).
Product Datasheet
Product Datasheet
Selecting Proper Controls
Selecting Proper Controls
Introduction
It is important to know if and when to use the proper controls for this product. The reporter lentivirus behavior will depend on the situation and therefore vary based on the conditions and cells it is being used in. We sell positive and negative control lentiviral particles which can be helpful in optimizing the conditions necessary to activate or inhibit the reporter activity, normalize luminescent signal (i.e. Renilla internal control), and troubleshoot the needed transduction conditions. Not everyone will need to use these controls, particularly if they are experienced with handling lentiviral reporters or perhaps if they have already purchased our product and determined ideal conditions for it already. However, we recommend using the proper controls always to ensure that the user can properly assess what they are seeing in their cells after transducing with our lentivirus. It should be noted that in some cases, an agonist (for example, TNF-alpha or LPS) may be needed to activate the reporter, and not all commercially-available agonists will work. We have had instances where TNF-alpha from one company cannot activate one of our reporters, whereas seemingly the same product from another company can. In these instances, we will do our best to inform of this on the website, however it is ultimately the responsibility of the buyer to figure these things out. If you observe something like this, we encourage you to contact us so that we can make the information available to others on our website.
The bottom line: When in doubt, use controls!
There are several different kinds of controls that you should consider when purchasing a reporter lentivirus from us. You have to decide which ones you will need, since it may depend on your particular application. Below is a general description which should help you in determining your individual needs. If you have questions, you can just contact us.
I. The Actual TF Reporter
Cat. No.: Various
Purpose: Experimental. This is the actual reporter that you will use to test the transcriptional activity of the indicated response elements in your experimental conditions. Example: test if a condition results in activation or inhibition of NFκB transcriptional activity.
Explanation of components:
Selection: Refers to the drug selection marker gene that is used to allow you to select the transduced cells and make a stable cell line. Currently you get 2 options, either puro or BSD.
Enhancer: This refers to an optimized minimal enhancer from the human CMV promoter. It is present in all of our TF Reporter constructs and functions by amplifying the reporter transcription driven by specific TF binding to response elements and the minimal TATA promoter.
TF Binding: Refers to the specific transcriptional response elements that are present to allow pathway or TF specific activation of the reporter. They are generally arranged as tandem repeats and the number of repeats varies depending on the product.
TATA: Minimal TATA promoter. Alone, it is insufficient to drive strong transcription of the reporter. It works together with the TF-bound response elements to drive the reporter expression when the specific signal pathway or TF is transcriptionally active.
Reporter: The fluorescent or luminescent reporter that is used to readout the pathway or TF activity in the transduced cells. Currently you can choose GFP, RFP, or Firefly Luciferase (FLuc).
II. Controls
Purpose: Establish a frame of reference for “positive reporter signal” that is independent of the pathway or TF-specific transcriptional response elements. The construct is organized the same as the actual TF reporters, except that a synthetic “constitutively active” TRE sequence is used to drive reporter activation in the cells. The reporter signal in the resultant cells can be used as a positive control to gauge whether the transduction conditions need to be optimized or if your particular test conditions are insufficient to activate the actual TF reporter.
Example 1: You are trying a specific ligand to induce the activity of your TF reporter, and not seeing much reporter activation. The positive control can help you determine if this is an issue with your transduction or if the ligand you selected is unable to activate the reporter. You may need to troubleshoot the ligand, add another ligand, co-transfect the cells with another gene, or something else.
Example 2: You are testing the inhibitory effect of your compound on a specific signaling pathway and see that it produces good inhibition. You can use the reporter positive control cells to confirm the effect of the compound is specific to that signaling pathway, not an off-target effect such as broad inhibition of transcription or translation.
How to use: Transduce a separate batch of cells (not the same ones that will receive the actual TF reporter). The reporter positive control should have the same selection marker and reporter as the actual TF reporter you are using to assay the desired transcriptional activity. This ensures you can control for any impact of the drug selection. Treat the transduced cells the same way as the ones that were transduced with the TF reporter. Use the reporter signal you observe to get an idea what reporter activation should look like in your cells.
Purpose: Establish a baseline for any background reporter activity.
How to use: Transduce a separate batch of cells (not the same ones that will receive the actual TF reporter). The reporter negative control should have the same selection marker and reporter as the actual TF reporter you are using to assay the desired transcriptional activity. This ensures you can control for any impact of the drug selection. Treat the transduced cells the same way as the ones that were transduced with the TF reporter. Any reporter activation or inhibition you observe represents the basal level (independent of transcriptional response elements).
RLuc Internal Control (Constitutive)
Purpose: Establish a frame of reference for “positive reporter signal” that is independent of the pathway or TF-specific transcriptional response elements. The advantage of the RLuc internal control is that it can be used in the same cells that receive the actual TF reporter construct driving Firefly luciferase. Thus, it can be in dual luciferase assays. The construct is organized the same as the actual TF reporters, except that a synthetic “constitutively active” TRE sequence is used to drive reporter activation in the cells. The reporter signal in the resultant cells can be used as a positive control to identify well-to-well variations since the Renilla luciferase can be detected separately from Firefly luciferase in the same batch of cells.
Example 1: You are trying a specific ligand to induce the activity of your TF reporter, and not seeing much Firefly luciferase reporter activation. If Renilla luc is also not being detected, it could indicate an issue with your cells, transduction/selection conditions, the instrument, or specific reagents being used. If you can detect RLuc but not FLuc, then it likely means the effect is pathway or TF-specific based on the TF reporter you are using. Note: to rule out reagent- or equipment-specific issues related to FLuc but not RLuc, you would need to transduce a separate batch of cells with Reporter Positive Control (LTV-0072) with FLuc as the reporter. The positive control can help you determine if this is an issue with your transduction or if the ligand you selected is unable to activate the reporter. You may need to troubleshoot the ligand, add another ligand, co-transfect the cells with another gene, or something else.
Example 2: You are testing the inhibitory effect of your compound on a specific signaling pathway and see that it produces good inhibition. You can use the constitutive RLuc internal control to verify that the effect of the compound is specific to that signaling pathway, not an off-target effect such as broad inhibition of transcription or translation.
How to use: Transduce the same batch of cells that will receive or already received the actual TF reporter. The RLuc Internal Control (Constitutive) should NOT have the same selection marker as the actual TF reporter you are using to assay the desired transcriptional activity. For example, if the actual TF reporter is using puro for selection, then the RLuc internal control should use blasticidin (BSD) and vice versa. This ensures you can select the same cells twice, once for the actual TF reporter, and again for the internal control. We strongly recommend that this be performed in two separate transductions. You should transduce the cells with one of the lentiviruses first, select the transduced cells, and let them grow up and completely recover before proceeding to the next round of transduction and selection with the second lentivirus. Details can be found in our transduction protocol which comes with each product and is also available under a different tab on this page.
RLuc Internal Control (Minimal)
Purpose: Establish a baseline for any background reporter activity in the same cells that contain the actual TF reporter construct. The advantage of the RLuc Internal Control is that it can be used in the same cells that receive the actual TF reporter construct driving Firefly luciferase. Thus, it can be used in dual luciferase assays. The construct is organized the same as the actual TF reporters, except that there are no transcriptional response elements to drive the expression of RLuc. Thus, any RLuc signal that is produced under your experimental conditions represents the basal activity of the construct independent of the transcriptional response elements (minimal TATA and upstream enhancer). This is useful in experiments where you are trying to study the reporter activation and need know the background level in dual-luciferase assays.
How to use: Transduce the same batch of cells that will receive or already received the actual TF reporter. The RLuc Internal Control (Minimal) should NOT have the same selection marker as the actual TF reporter you are using to assay the desired transcriptional activity. For example, if the actual TF reporter is using puro for selection, then the RLuc internal control should use blasticidin (BSD), and vice versa. This ensures you can select the same cells twice, once for the actual TF reporter, and again for the internal control. We strongly recommend that this be performed in two separate transductions. You should transduce the cells with one of the lentiviruses first, select the transduced cells, and let them grow up and completely recover before proceeding to the next round of transduction and selection with the second lentivirus. Details can be found in our transduction protocol which comes with each product and is also available under a different tab on this page.