NFκB-TIME (ATCC® CRL-4049)

Organism: Homo sapiens, human  /  Cell Type: Microvascular endothelial cell  /  Tissue: foreskin  /  Disease: normal

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Organism Homo sapiens, human
Tissue foreskin
Cell Type Microvascular endothelial cell
Product Format frozen
Morphology endothelial
Culture Properties adherent
Biosafety Level 2  [Cells contain SV40 viral DNA sequences]
Disease normal
Age neonatal
Gender male
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.

Storage Conditions liquid nitrogen vapor phase
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.
Images
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.

Clinical Data male
neonatal
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.

Complete Growth 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.
Subculturing 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.
Cryopreservation 90% FBS, 10%DMSO
Culture Conditions Atmosphere: air, 95%; carbon dioxide (CO2), 5% 
Temperature: 37°C
STR Profile D5S818:  11            
D13S317: 9, 11
D7S820: 8, 9        
D16S539: 9, 12
vWA: 16, 18             
Amelogenin:  X, Y    
TPOX: 8
CSF1PO: 11, 12
TH01: 6, 7
Population Doubling Level (PDL) As part of our quality control, we have tested this cell line for its ability to grow for a minimum of 15 population doublings after recovery from cryopreservation. We have also compared its karyotype, telomerase expression level, growth rate, morphology and tissue-specific markers when first recovered from cryopreservation with that of cells at 10+ population doublings to ensure that there is no change in these parameters and that the cells are capable of extended proliferation.
Basic Documentation
Other Documentation
FAQ's
  1. Freezing NFkB-TIME cells
    We recommend freezing the NFkB-TIME cells in a solution of 90% FBS / 10% DMSO.
    Date Updated: 2/13/2014
  2. Subculture NFkB-TIME cells
    We recommend subculturing the NFkB-TIME, ATCC® CRL-4049 ™ cells when they reach about 80% to 90% confluence.
    Date Updated: 2/13/2014
  3. Antibiotics in NFkB-TIME culture medium
    The culture medium for ATCC® CRL-4049 ™, NFkB-TIME cells should be supplemented with both blasticidin and hygromycin to ensure continued expression of hTERT and the NanoLuc® lu...
    Date Updated: 2/13/2014
  4. Optimal seeding density for NFkB-TIME cells

    The optimal seeding density for NFkB-TIME, ATCC® CRL-4049 ™ cells is from 4 - 6 x 103 cells/cm2.


    Date Updated: 2/12/2014
  5. NFkB-TIME culture medium


    Date Updated: 2/13/2014

  6. NFkB-TIME tube formation
    We recommend using CellMatrix Basement Membrane Gel (ATCC® ACS-3035 ™) for the tubulogenesis assay.  In addition, the number of NFkB-TIME cells seeded onto the basement membrane is...
    Date Updated: 2/13/2014
  7. Primary vs. immortalized microvascular endothelial cells
    Primary microvascular endothelial cells may grow for 15 population doublings (PD) before they become senescent and undergo growth arrest.  The hTERT-immortalized microvascular cell lines,&nb...
    Date Updated: 2/13/2014
  8. Difference between microvascular endothelial growth kits

    Your experimental design will dictate which microvascular endothelial cell growth kit should be used.


    Date Updated: 2/13/2014
  9. Thawing NFkB-TIME cells

    Thaw the vial by gentle agitation in a 37°C water bath for approximately 2 minutes.


    Date Updated: 2/13/2014
  10. Measure NanoLuc® luciferase activity


    Date Updated: 2/24/2014

Restrictions

This material is subject to claims under U.S. Patent Nos. 6,261,836 and 6,337,200, other pending patent applications, and foreign counterparts thereof. It is required that either the Addendum for Commercial and For-Profit Organizations or the Addendum for Noncommercial and Academic Organizations, as appropriate, be signed and returned to ATCC before shipment. 

In addition to the foregoing, this product's use is governed by a Limited License. For information on purchasing a license to use this product for purposes other than those permitted in the Label License, please contact Promega.

In addition to the foregoing, any proposed commercial use of ATCC® CRL-4049 NFκB-TIME must also be negotiated with University of California, San Francisco, Office of Technology Management, 3333 California Street, Suite S-11, San Francisco, CA 94143-1209, Tel: 415.502.1585, Fax: 415.502.1661.