KC-5700

Hep3B-mouse-STEAP2-ECD Cell Line

59326

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Background of Hep3B-mouse-STEAP2-ECD Cell Line

This gene is a member of the STEAP family and encodes a multi-pass membrane protein that localizes to the Golgi complex, the plasma membrane, and the vesicular tubular structures in the cytosol. A highly similar protein in mouse has both ferrireductase and cupric reductase activity, and stimulates the cellular uptake of both iron and copper in vitro. Increased transcriptional expression of the human gene is associated with prostate cancer progression. Alternate transcriptional splice variants, encoding different isoforms, have been characterized.

Specifications

Catalog Number	KC-5700
Cell Line Name	Hep3B-mouse-STEAP2-ECD Cell Line
Clone Number	6#
Host Cell Line	Hep3B
Description	Stable Hep3B cell line expressing exogenous mouse-STEAP2-ECD 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 1:2-1:3 every 3-4 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 40 hours
Mycoplasma Status	Negative

Cell Line Generation

Hep3B-mouse-STEAP2-ECD cell line was generated using a lentiviral vector expressing the mouse-STEAP2-ECD sequence.

Characterization

Figure 1: Characterization of mouse-STEAP2-ECD overexpressing in Hep3B-mouse-STEAP2-ECD stable clone using FACS.

Figure 2: Characterization of mouse-STEAP2-ECD overexpressing in Hep3B-mouse-STEAP2-ECD stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 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 1:2-1:3 every 3-4 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

Whiteland H, Spencer-Harty S, Morgan C, Kynaston H, Thomas DH, Bose P, Fenn N, Lewis P, Jenkins S, Doak SH. A role for STEAP2 in prostate cancer progression. Clin Exp Metastasis. 2014 Dec;31(8):909-20. doi: 10.1007/s10585-014-9679-9. Epub 2014 Sep 24. PMID: 25248617.
Burnell SEA, Spencer-Harty S, Howarth S, Bodger O, Kynaston H, Morgan C, Doak SH. STEAP2 Knockdown Reduces the Invasive Potential of Prostate Cancer Cells. Sci Rep. 2018 Apr 19;8(1):6252. doi: 10.1038/s41598-018-24655-x. PMID: 29674723; PMCID: PMC5908900.