KC-6739

Hepa1-6-puro Cell Line

66381

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Background of Hepa1-6-puro Cell Line

Puromycin is an aminoglycoside antibiotic isolated from the bacterium Streptomyces alboniger. It inhibits protein synthesis by acting as an analog of the 3’ terminal end of aminoacyl-tRNA. During translation, it incorporates into the growing polypeptide chain at the ribosomal A site, causing premature termination and the release of an incomplete peptide. This agent exhibits antimicrobial, antitrypanosomal, and antineoplastic properties and is widely used in molecular biology research as a selection agent in cell culture to establish stable cell lines expressing the puromycin resistance gene (pac gene).

Specifications

Catalog Number	KC-6739
Cell Line Name	Hepa1-6-puro Cell Line
Host Cell Line	Hepa1-6
Description	Hepa1-6 cell line stably expressing exogenous puro gene
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM+10%FBS+2μg/mL puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

Hepa1-6-puro cell line was generated using a lentiviral vector expressing the puro sequence.

Characterization

Cell Resuscitation

Prewarm culture medium (DMEM+10%FBS+2μg/mL puromycin) in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:4-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage.

References

Nathans D, et al. Puromycin inhibition of protein synthesis: incorporation of puromycin into peptide chains. Proc Natl Acad Sci U S A. 1964 Apr;51:585-92.
Schmidt EK, et al. SUnSET, a nonradioactive method to monitor protein synthesis. Nat Methods. 2009 Apr;6(4):275-7.
3. Shao YG, et al. Podocin expression in rats with puromycin aminonucleoside nephropathy. Zhongguo Dang Dai Er Ke Za Zhi. 2004 Jun;6(3):161-5.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.