Jurkat-Dual™ Cells
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Cat.code:
jktd-isnf
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ABOUT
IRF-SEAP & NF-κB-Luc Reporter T Lymphocytes
Jurkat-Dual™ cells are engineered reporter T lymphocytes designed for the study of transcription factor activation in a physiologically-relevant cell line. These cells stably express two inducible reporter constructs; interferon regulatory factor-inducible secreted embryonic alkaline phosphatase (IRF-SEAP) and NF-κB-Lucia luciferase (NF-κB-Luc) reporter genes. They were derived from the human T lymphocyte-based Jurkat cell line.
Cell line description:
Jurkat-Dual™ cells feature the Lucia luciferase gene, a secreted luciferase reporter gene, driven by an interferon-β (IFN-β) minimal promoter fused to five copies of the NF-κB consensus transcriptional response element and three copies of the c-Rel binding site. These cells also express a secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of an IFN-stimulated gene 54 (ISG54) minimal promoter in conjunction with five IFN-stimulated response elements. As a result, Jurkat-Dual™ cells allow the simultaneous study of the NF-κB pathway, by monitoring the activity of Lucia luciferase, and the IRF pathway, by assessing the activity of SEAP. Both reporter proteins are readily measurable in the cell culture supernatant when using QUANTI-Luc™ 4 Lucia/Gaussia, a Lucia and Gaussia luciferase detection reagent, and QUANTI-Blue™ Solution, a SEAP detection reagent.
Jurkat-Dual™ cells induce the activation of NF-κB in response to TNF-α and T-Lymphocyte mitogens, such as phytohemagglutinin and concanavalin A. They trigger the IRF pathway upon stimulation with type I IFNs and poly(I:C).
Features of Jurkat-Dual™ cells:
- Readily assessable SEAP and Lucia luciferase reporter activity
- The stability for 20 passages has been verified
- Functionally tested and guaranteed mycoplasma-free
Application of Jurkat-Dual™ cells:
- Monitoring IRF and NF-κB activation in T lymphocytes
Disclaimer: These cells are for internal research use only and are covered by a Limited Use License (See Terms and Conditions). Additional rights may be available.
SPECIFICATIONS
Specifications
Screening of PRR agonists or inhibitors
Complete IMDM (see TDS)
Verified using Plasmotest™
Each lot is functionally tested and validated.
CONTENTS
Contents
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Product:Jurkat-Dual™ Cells
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Cat code:jktd-isnf
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Quantity:3-7 x 10^6 cells
- 1 ml of Blasticidin (10 mg/ml)
- 1 ml of Zeocin® (100 mg/ml)
- 1 ml of Normocin™ (50 mg/ml)
- 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)
- 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)
Shipping & Storage
- Shipping method: Dry ice
- Liquid nitrogen vapor
Storage:
Details
The human Jurkat cell line was established from acute T cell leukemia. Jurkat cells have been extensively used in vitro to delineate the signaling pathways induced by the engagement of T-cell receptors and to study the expression of various chemokine receptors susceptible to viral entry, particularly HIV [1]. T cell activation results in the nuclear translocation of transcription factors, such as IRFs (interferon regulatory factors) and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) to induce the expression of target genes. Knowledge of their signaling cascades, ease of culture, and transfection make Jurkat cells a convenient tool to screen for T-cell activation, anti-viral or anti-cancer drugs.
References:
1. Montano M. 2014. Model systems. Translational Biology in Medicine. 9-33.
DOCUMENTS
Documents
Technical Data Sheet
Safety Data Sheet
Validation Data Sheet
Certificate of analysis
Need a CoA ?
You may also need
FAQ
Handling of cells upon arrival
In general, InvivoGen’s cells are shipped on dry-ice and since dry-ice is not nearly as cold as liquid nitrogen, thawing of the cells technically begins during transport. Thus, we recommend a full thaw upon receipt to ensure the best recovery results.
NOTE: this is not applicable in some countries in Asia where cell lines are sent at room temperature using our specially designed flask
The most important step is the thawing procedure upon receipt of the cells.
Do not put your cells at -80°C or in liquid nitrogen as this may damage the cells.
You must propagate the cells immediately upon receipt.
Below are a few tips we recommend if you encounter any issues during initial culture:
• For the first 2-3 passages, grow cells in media containing 20% FBS and no antibiotics.
• Do not allow cells to reach 100% confluency. Please check the cells as regularly as possible.
• The cells should not be grown in 20% FBS for too long. Use media with 10% FBS after 2 or 3 passages.
• When making frozen stocks, continue growing additional cultures in case there is a problem with the frozen stock.
If you are working with THP1 cells in particular, here are a few additional tips:
• THP1 cells need to be passaged between 3 x 105 and 7 x 105 cells per mL (we recommend 5 x 105 cells per ml). Below this, your cells will take a long time to grow and above 2 x 106 cells per ml you may have toxicity.
• Between each passage, do not centrifuge the cells. THP1 cells grow better in conditioned media, therefore when passaging cells, leave 1 – 2 ml of the old media and add fresh media on top. Do not leave more than 50% of conditioned media as the cells will not have enough nutrients to properly grow.
After thawing, the cells can be more sensitive to selective antibiotics due to the initial low levels of resistance markers. Therefore, it is recommended to wait for 2 - 3 passages before adding the selective antibiotics in order to avoid further stress to the cells during the first couple of passages.
Cell culture media
In house we inactivate serum by heating at 56°C for 30 minutes. It is important to wait for the water bath to reach the 56°C before placing the tube of serum in to ensure the serum is fully inactivated.
In house we always use heat-inactivated serum for the freezing medium, growth medium and test medium. However, please note that it is not a problem if you wish to use non-heat-inactivated in the freezing medium.
It is important to choose an endotoxin-free serum for the culture of our reporter cell lines. InvivoGen systematically asks for the Certificate of Analysis of different batches of serum to ensure a low level of endotoxin as to not activate the NFkB pathway.
100 U/ml Penicillin and 100 µg/ml Streptomycin
No, this shouldn’t be a problem as long as there is at least 2 mM L-Glutamine in the medium. Most DMEM formulations already contain 4mM of L-Glutamine. However, if you need to add L-Glutamine to your medium you will only need to add 2 mM for the cells to grow properly.
Both Glutamine and GlutaMax have been tested in-house and can be used, with no difference in the cells noted.
Many of InvivoGen’s cell lines are engineered with a SEAP (Secreted Embryonic Alkaline Phosphatase) reporter system, and FBS, in many cases, contains traces of alkaline phosphatase (AP) that interferes with the test results. Therefore, to avoid background noise we recommend working with inactivated serum with all of our cells.
Cell line culture
When the HEK-Blue™ cells are non-adherent, either they were diluted too harshly at the start or they have grown over-confluent in a small flask and suffocated.
To avoid this in the future:
• Change the medium and seed the cells at a density of approximately 1.5 x 106 cells/mL in a T25 flask.
• Wash the cells before putting them into a new flask. Sometimes when the cells are non-adherent, it is due to the clustering of both live and dead cells. Therefore, this will get rid of any remaining DMSO which could affect the adhesion of the cells to the flask.
• Use medium with 20% FBS.
• The use of CellBIND flasks can sometimes help to increase attachment and growth of the cells (however CellBIND flasks are not required in the normal protocol)
It isn’t normal for your cells to divide only once a week. Depending on the clone, they usually have a doubling time of 24 – 72 hours. THP1 cells must be cultured at quite a high concentration (at least 5 x 105 cells/ml). Please note that our R&D teams have noticed that THP1 cells often die when they are diluted too harshly. Also, it may help to not add Normocin™ while waiting for improvement to their growth.
It is not a problem if the cells are clumping, as long as they are growing fine and have normal morphology. You may want to centrifuge the cells to get rid of the dead cells as sometimes this is the reason for clumping. Please note that we recommend homogenizing the cells before performing your assays.
We recommend to centrifuge at 120 G for 10 minutes.
Assays
They are semi-quantitative tests. However, they can be used for quantitative measurements by making a standard curve using a positive control.
If you cannot run the detection assays immediately, you can store supernatant samples for up to a week at 4°C. Supernatant samples are active for a longer time when kept at -20°C. However, in this case we would recommend supplementing the test medium with 20% FBS to protect the SEAP as much as possible during the freezing process. Do not repeat freeze/thaw cycles.
It depends on the inhibitor. If the inhibitor blocks a receptor or an element in the signaling pathway, we recommend pre-incubating the inhibitor with the cells for at least 30 minutes. If the inhibitor is binding or blocking the ligand (i.e. an antibody against a specific cytokine) then it is best to pre-incubate the inhibitor with the ligand prior to adding to the cells.
In house the cells are seeded at the same time as adding the ligand (ligand first).
To limit the activation of NFκB before stimulation (lower background):
• Use healthy cells that have been passaged at least 24 hours before the assay
• Do not allow cells to be greater than 80 % confluent
• Use pre-warmed PBS to rinse your cells
• Use heat-inactivated FBS (to eliminate residual alkaline phosphatase in the serum)
• Do not centrifuge the cells prior to stimulation
• Rather than trypsin, use PBS for 2 - 3 min @ 37°C and gently pipet up and down to detach cells
• Do not use an excessive number of cells per well
(approximately 50 000 cells/well as recommended on the TDS).
We recommend to not use Normocin™ in the test medium with all of our cell lines as it is better to reduce the number of potential interfering agents in the medium when performing the assay. The same goes for the use of selective antibiotics
It is hard to estimate the deviation factor regarding cell passage number. However, we note very little difference in our experimental results, with no more than the slight variations you expect due to handling errors.
Yes, InvivoGen’s reporter cell lines can be used in ELISA and Western Blots. However, please note that we do not sell them for this purpose.
Frozen stock preparation
For adherent cells, we recommend DMEM, 20% (v/v) fetal bovine serum (FBS), and 10% (v/v) DMSO.
For suspension cells, we recommend fetal bovine serum (FBS) and 5-10% (v/v) DMSO
We highly recommend keeping a flask of cells running until you begin thawing your frozen stock in case something has gone wrong during the freezing step.
Reporter system
Our reporter cells require the activation of just one transcription factor (NFκB and/or AP-1) to produce a signal.
InvivoGen’s Lucia® luciferase is completely synthetic and is not related to any natural luciferase.