TIME-GFP (ATCC® CRL-4045)

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

Permits and Restrictions

View Permits View Restrictions

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 TIME-GFP (ATCC CRL-4045) and its parental cell line TIME (ATCC CRL-4025) exhibit normal diploid karyotype, extended lifespan in culture and endothelial characteristics that make them an ideal model for investigating many aspects of endothelial cell biology. TIME-GFP (ATCC CRL-4045) retains angiogenic potential and stably expresses green fluorescence protein (GFP) for at least 12 passages. The GFP-expressing cells migrate and coalesce into networks of vessel-like structure within 8 hours after being plated onto basement membrane gel (CellMatrix, ATCC ACS-3035). The stable expression of GFP in these cells enables detection and analysis of the fragile endothelial structures to occur without post-assay fixation and/or staining.
Shipping Information FZ
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 shows some karyotypic instability at later passages.
Images
Derivation TIME-GFP (ATCC CRL-4045) is derived by transfecting TIME (ATCC CRL-4025) cells with linearized pWE2-EmGFP plasmid, which constitutively expresses the Green Fluorescence Protein (GFP) driven by a CMV promoter.   A stable GFP-expressing clone is selected to establish the TIME-GFP cell line.
Clinical Data male
neonatal
Antigen Expression Positive for CD31 and capable of uptaking Low Density Lipoprotein (LDL). Express Green Fluorescence Protein (GFP) constitutively
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 ug/mL and G418 (Life Technologies Cat. No. 11811-023) at a final concentration of 200 ug/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.
Name of Depositor ATCC
Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
  • License agreement required for commercial customer uses.
  • This material is distributed for research purposes only. A signed addendum to the ATCC Material Transfer Agreement must be sent to ATCC in advance of shipment.
Basic Documentation
Other Documentation
FAQ's
  1. TIME-GFP subculture
    Subculture the TIME-GFP cells when the culture reaches 80-90% confluency.
    Date Updated: 3/27/2014
  2. TIME-GFP tubulogenesis assay
    CellMatrix Basement Membrane Gel (ATCC® ACS-3035 ™) is recommended for the tubulogenesis assay.  In addition, the number of TIME-GFP cells seeded onto CellMatrix is critical. ...
    Date Updated: 3/27/2014
  3. TIME-GFP seeding density
    Optimal seeding density for TIME-GFP cells is 4 x 10
    Date Updated: 3/27/2014
  4. TIME-GFP culture medium
    Vascular Cell Basal Medium (ATCC® PCS-100-030 ™) supplemented with one of the following microvascular cell growth kits: Microvascular Endothelial Cell Growth Kit-BBE (ATCC® PCS-110-...
    Date Updated: 3/27/2014
  5. TIME-GFP growth kit selection

    Your experimental design will dictate which Endothelial Cell Growth Kit should be used.


    Date Updated: 3/27/2014
  6. TIME-GFP antibiotics in medium
    The medium for TIME-GFP (ATCC® CRL-4045 ™) should be supplemented with blasticidin and G418 to ensure expression of the hTERT and GFP proteins, respectively.  Add blastic...
    Date Updated: 3/27/2014
  7. TIME-GFP expression of VEGFR-2 and Tie-2
    VEGFR-2 (KDR/Flk-1) appears to mediate almost all of the known cellular responses to VEGF.  Tie-2, also named TEK Tyrosine Kinase, is involved in vascular stabilization and remodeling. ...
    Date Updated: 3/27/2014
  8. Difference between primary and hTERT immortalized microvascular cell lines

    Primary microvascular endothelial cells may grow for 15 population doublings (PD) until they become senescent and undergo growth arrest.


    Date Updated: 3/27/2014
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 the Limited Use License.  For information on purchasing a license to use this product for research (for-profit entities) or commercial purposes (any entity) other than those permitted in the limited use label license, contact the Licensing Department, Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, California 92008. Phone (760) 603-7200 or outlicensing@lifetech.com

In addition to the foregoing, any proposed commercial use of ATCC® CRL-4045™ [TIME-GFP] 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.