KC-6331

LNcap C4-2B-STEAP2-KO cell line

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Background of LNcap C4-2B-STEAP2-KO cell line

Six transmembrane antigen of prostate 2 (STEAP2) also known as the six transmembrane protein of prostate 1 (STAMP1) is a member of the metalloreductase family important in the metal reduction of copper and iron. In vitro and in vivo studies show that STEAP2 plays a key role in prostate cancer progression. STEAP2 is located on the plasma membrane of prostate cells and Golgi complex. It increases prostate cancer progression, controls cell proliferation, differentiation, and decreases apoptosis. Its knockdown from prostate cancer cells has been shown to reduce their invasive potential, increased apoptosis, and reduced migration that are responsible for oncogenesis and disease progression. Immunohistochemical staining significantly demonstrates its expression at the cell–cell junctions of prostate cancer cells. It is differentially expressed in normal and cancerous tissue making it a potential target for new therapeutic strategies for disease treatment. STEAP2 is expressed more than 10 times in normal prostate than in other tissues such as the brain and liver and is exponentially expressed in malignant prostate cancer cells. Their levels in these tissues are too low to have any functional significance. STEAP2 is highly expressed at all stages of prostate cancer and is androgen independent, a characteristic that is key in managing androgen-dependent and independent/advanced prostate cancer. Its unique and specific upregulation in cancerous prostate tissue at all stages is likely to make it an ideal therapeutic drug target.

Specifications

Catalog Number	KC-6331
Cell Line Name	LNcap C4-2B-STEAP2-KO cell line
Clone Number	1C1
Host Cell Line	LNcap C4-2B
Description	Stable LNcap C4-2B-STEAP2-KO cell clone with human STEAP2 gene knockout
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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	Waymouth’s MB752/1+10%FBS+2mM Glutamine/RPMI1640+10%FBS+2mM Glutamax
Selection Marker	NA
Morphology	Fibroblastoid cells growing as a monolayer
Subculture	Split the saturated culture at a ratio of 1:3-1:4 every 3-4 days; seed out at about 1-3 x 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 36 hours
Mycoplasma Status	Negative

Cell Line Generation

LNcap C4-2B-STEAP2-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of LNcap C4-2B-STEAP2-KO cell line stable clone using PCR sequencing.

Figure 2:Characterization of LNcap C4-2B-STEAP2-KO cell line stable clone using RT-PCR sequencing.

Figure 3:Characterization of LNcap C4-2B-STEAP2-KO cell line stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (Waymouth’s MB752/1+10%FBS+2mM Glutamine/RPMI1640+10%FBS+2mM Glutamax)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 1:3-1:4 every 3-4 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Ongaba T, Ndekezi C, Nakiddu N. A Molecular Docking Study of Human STEAP2 for the Discovery of New Potential Anti-Prostate Cancer Chemotherapeutic Candidates. Front Bioinform. 2022 May 24;2:869375. doi: 10.3389/fbinf.2022.869375. PMID: 36304279; PMCID: PMC9580961.