Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

The cell line appears to be derived from a single cell based on Southern and FISH analysis.

adult

male

epidermal growth factor (EGF), expressed

alkaline phosphatase; gamma glutamyltranspeptidase; leucine aminopeptidase; acid phosphatase; cytokeratin; alpha 3, beta 1 integrin; fibronectin

alkaline phosphatase; gamma glutamyltranspeptidase; leucine aminopeptidase; acid phosphatase; cytokeratin; alpha 3, beta 1 integrin; fibronectin

The recombinant retrovirus vector pLXSN 16 E6/E7 containing the HPV-16 E6/E7 genes was used to transfect the ectotropic packaging cell line Psi-2.

Virus produced by the Psi-2 cells was used to infect the amphotropic packaging cell line PA317 (see ATCC CRL-9078).

Virus produced by the PA317 cells was used to transduce primary PTCs.

Although pLXSN 16 E6/E7 also confers resistance to neomycin, selection in G418 was not used to isolate transduced clones.

The cell line appears to be derived from a single cell based on Southern and FISH analysis.

The E6/E7 genes are present in the HK-2 genome as determined by PCR.

The cells retain a phenotype indicative of well differentiated PTCs.

They are positive for alkaline phosphatase, gamma glutamyltranspeptidase, leucine aminopeptidase, acid phosphatase, cytokeratin, alpha 3,beta 1 integrin, and fibronectin.

The cells are negative for factor VIII related antigen, 6.19 antigen and CALLA endopeptidase.

HK-2 cells retain functional characteristics of proximal tubular epithelium such as Na+ dependent / phlorizin sensitive sugar transport and adenylate cyclase responsiveness to parathyroid, but not to antidiuretic hormone.

The cells are capable of gluconeogenesis as evidenced by their ability to make and store glycogen.

HK-2 cells are anchorage dependent.

The cells will not grow in methylcellulose, soft agar or suspension.

HK-2 cells can reproduce experimental results obtained with freshly isolated PTCs.

Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.

Note: The cells should not be allowed to become confluent, subculture at 80% of confluence.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.05% (w/v) Trypsin- 0.53 mM EDTA solution.
3. Add 2.0 to 3.0 mL of 0.05% Trypsin-0.53 mM 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¡ãC to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels.
6. Incubate cultures at 37¡ãC.

Subcultivation Ratio: A subcultivation ratio of 1:4 is recommended

Medium Renewal: Every 2 to 3 days

Freeze medium: Complete growth medium supplemented with 7.5% (v/v) DMSO

Storage temperature: liquid nitrogen vapor phase

Atmosphere: air, 95%; carbon dioxide (CO2), 5%

Temperature: 37¡ãC

Growth Conditions: Cell growth is dependent on epidermal growth factor. The cells should not be allowed to become confluent. Subculture at 80% of confluence.