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NFκB-TIME

CRL-4049

Product category
Human cells
Product type
hTERT-immortalized cell
Organism
Homo sapiens, human
Cell type
microvascular endothelial cell
Morphology
endothelial
Tissue
Skin; Foreskin
Disease
Normal
Applications
3D cell culture
Cardiovascular disease research
Drug development
High-throughput screening
Product format
Frozen
Storage conditions
Vapor phase of liquid nitrogen
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Documentation

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

Cells contain SV40 sequences

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Detailed product information

General

Specific applications

Both NFkB-TIME (CRL-4049) and its parental cell line TIME (CRL-4025) exhibit normal diploid karyotype, extended lifespan in culture and endothelial characteristics that make them ideal models for investigating many aspects of endothelial cell biology.  



NFkB-TIME (ATCC CRL-4049) retains angiogenic potential and the expression of NanoLuc® luciferase under the control of multiple copies of the NFkB response element.  When the NFkB-TIME (ATCC CRL-4049) cells are exposed to inflammatory cytokine such as TNFα, activation of the NFkB signaling pathway occurs resulting in increased NanoLuc® luciferase activity.  The high sensitivity, excellent signal/background ratio and simple single-addition assay makes this reporter cell line an ideal replacement for the cumbersome and highly variable ICAM-1/CD54 activation assays.

Characteristics

Growth properties
Adherent
Derivation

TIME (ATCC CRL-4025) cells are transfected with linearized pNL3.2.NF-κB-RE[NlucP/NF-κB-RE/Hygro] plasmid, which expresses the NanoLuc® luciferase under the control of multiple copies of the NFkB response element.   A stable clone with high activation of the NanoLuc® luciferase activity upon exposure to inflammatory cytokine TNFα is selected to establish the NFkB-TIME cell line.

Age
neonate
Gender
Male
Immortalization method
hTERT expression
Karyotype
This is a diploid cell line of male origin with a modal chromosome number of 46 and a low rate of polyploidy. The line may show some karyotypic instability at later passages.
Antigen expression
Positive for CD31 and capable of uptaking Low Density Lipoprotein (LDL).  
Express NanoLuc® luciferase under the control of multiple copies of the NFkB response element.
Comments

TIME (ATCC CRL-4025) cells are transfected with linearized pNL3.2.NF-κB-RE[NlucP/NF-κB-RE/Hygro] plasmid, which expresses the NanoLuc® luciferase under the control of multiple copies of the NFkB response element.   A stable clone with high activation of the NanoLuc® luciferase activity upon exposure to inflammatory cytokine TNFα is selected to establish the NFkB-TIME cell line.

Handling information

Unpacking and storage instructions
  1. Check all containers for leakage or breakage.
  2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.
Complete medium
The base medium for this cell line is Vascular Cell Basal Medium (ATCC PCS-100-030), supplemented with Microvascular Endothelial Cell Growth Kit-BBE (ATCC PCS-110-040) OR Microvascular Endothelial Cell Growth Kit-VEGF (ATCC PCS-110-041). Add Blasticidin (Life Technologies Cat. No. A11139-03) at a final concentration of 12.5 µg/mL and Hygromycin (Life Technologies Cat. No. 10687-010) at a final concentration of 20 µg/mL.

Note: Do not filter complete medium.
Temperature
37°C
Atmosphere
95% Air, 5% CO2
Handling procedure
To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt.
If storage of the frozen culture is necessary upon arrival, store the vial in liquid nitrogen vapor phase and NOT
at –70°C. Storage at –70°C will result in loss of viability.
  1. Prepare a 25 cm2 or a 75 cm2 culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents, the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
  2. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
  3. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All operations from this point on should be carried out under strict aseptic conditions.
  4. Transfer the vial contents to a centrifuge tube containing 9.0 mL of complete culture medium and centrifuge the cell suspension at approximately 125 x g for 5 to 7 minutes.
  5. Discard the supernatant and resuspend the cells in fresh growth medium (see the batch-specific information for the recommended dilution ratio). Add this suspension to the prepared culture vessel.
  6. Incubate the culture at 37°C in a suitable incubator.
Subculturing procedure
Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation solutions for culture vessels of other sizes.

Subculture when the culture is about 90% confluent.
  1. Remove and discard spent medium.
  2. Briefly rinse the cells with Dulbecco's Phosphate Buffered Saline (DPBS, ATCC 30-2200) and discard rinse solution.
  3. Add 2.0 to 3.0 mL room temperature Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) to the flask. Incubate at 37°C for 5 min (until cells have detached).
  4. Neutralize trypsin by adding an equal volume of room temperature 2% FBS in DPBS.
  5. Centrifuge cells at 250 x g for 5 min at room temperature.
  6. Remove supernatant. Resuspend pellet in 6.0 to 8.0 mL Complete Growth Medium.
  7. Count cells, and seed 5.0 x 103 to 8.0 x 103 viable cells/cm2 to new culture vessels.
Medium Renewal: Every 2-3 days.
Reagents for cryopreservation
Fetal bovine serum supplemented with 10% (v/v) DMSO (ATCC 4-X)

Quality control specifications

Population doubling capacity
≥ 15 in complete growth medium
STR profiling
D5S818:  11            
D13S317: 9, 11
D7S820: 8, 9        
D16S539: 9, 12
vWA: 16, 18             
Amelogenin:  X, Y    

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

Material Transfer Agreement Addendum for Screening Applications

For-profit organizations
For every order of this item, you must provide a signed Material Transfer Agreement Addendum for Screening Applications. We cannot ship this item until we receive this addendum. The person signing the addendum as the principal investigator must match the end user as listed on the applicable sales order for the item.

Email the signed addendum to [email protected] with a reference to both your account and sales order numbers. Once received, your addendum will be reviewed, and this item will be released for shipment if all requirements are met. Additional fees may apply if this product is being used for a screening use (ATCC ACS-2103F), and these fees will be applied after your order is confirmed. If you need assistance with your order, please contact our Customer Care team or your applicable distributor.

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

This material is subject to the following restrictions in addition to those outlined in the ATCC Material Transfer Agreement:

  1. NanoLuc Label License 

For information on obtaining additional rights, please contact:

ATCC Licensing
Email: [email protected]

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

Frequently Asked Questions

References

Need assistance with this product? Contact our Technical Support team.

Telephone

Telephone

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Outside the US
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hours

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