Phoenix-AMPHO (ATCC® CRL-3213)

Organism: Homo sapiens, human  /  Tissue: kidney  / 

Organism Homo sapiens, human
Tissue kidney
Product Format frozen
Morphology epithelial
Culture Properties adherent
Biosafety Level 2 [Cells contain Adeno and SV-40 viral DNA sequences]
Age embryo
Applications

Phoenix is a second-generation retrovirus producer cell line for the generation of helper-free ecotropic and amphotropic retroviruses.

The unique feature of this cell line is that it is highly transfectable with either calcium phosphate mediated transfection or lipid-based transfection protocols-- up to 50% or higher of cells can be transiently transfected.

This cell line is capable of carrying episomes for long-term stable production of retrovirus (LZRS).

Storage Conditions liquid nitrogen vapor phase
Derivation HEK 293T/17 cells were transformed with adenovirus E1a carrying a temperature sensitive T antigen co-selected with neomycin. Transformation was brought about by the insertion of approximately 4.5 kilobases of viral genome into human chromosome 19.
Comments

Phoenix Amphotropic (AMPHO) cell line expresses amphotropic envelope protein.

CD8 expression is a direct reflection of intracellular gag-pol and the stability of the producer cell population's ability to produce gag-pol can be readily monitored by flow cytometry.

Phoenix-Ampho have been extensively tested for helper virus production and established as being helper-virus free.

 

Complete Growth Medium The base medium for this cell line is Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC 30-2002). To make the complete growth medium, add the following components to the base medium: 10% Fetal Bovine Serum (heat inactivated) (ATCC 30-2020), 2mM L-glutamine (ATCC 30-2214), 1% Penicillin/Streptomycin.
Subculturing
Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.
  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
  3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
    Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
  4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new culture vessels.
  6. Incubate cultures at 37°C.

Note:  Culture to ≤80% confluence to prevent the cells from sloughing

Subcultivation Ratio: A subcultivation ratio of 1:5 to 1:8 is recommended
Medium Renewal: Every 2 to 3 days

 

 

Cryopreservation Freeze medium:  complete growth medium supplemented with 5% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase
Culture Conditions Temperature: 37°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Cells per Vial 4.5 x 106 cells per vial
Population Doubling Time 16 hours
Name of Depositor G Nolan
Passage History This cell line was deposited at passage 10.
Year of Origin 1996
References

Swift S et al. Rapid Production of Retroviruses for Efficient Gene Delivery to Mammalian Cells Using 293T Cell-Based Systems. Current Protocols in Immunology,Unit 10.17C, 2001

Pear WS, et al. Production of High-Titer Helper-Free Retroviruses by Transient Transfection. Proc. Natl. Acad. Sci. USA 90: 8392-8396, 1993. PubMed: 7690960

Basic Documentation
References

Swift S et al. Rapid Production of Retroviruses for Efficient Gene Delivery to Mammalian Cells Using 293T Cell-Based Systems. Current Protocols in Immunology,Unit 10.17C, 2001

Pear WS, et al. Production of High-Titer Helper-Free Retroviruses by Transient Transfection. Proc. Natl. Acad. Sci. USA 90: 8392-8396, 1993. PubMed: 7690960