KC-4765

293T-cyno-STEAP1-Cell-Line

53544

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Background of 293T-cyno-STEAP1-Cell-Line

The STEAP family contains four members, named STEAP1–4, all of which have in common a six transmembrane domain with the COOH- and N-terminals located in the cytosol. STEAP1 was the first member of the STEAP family to be identified and has been widely studied as a gene related to cancer progression. STEAP1 was previously predicted not to promote iron and cooper reduction or uptake mainly due to the lack of the N-terminal NADPH-binding F420H2: NADP+ oxidoreductase domain unlike other STEAP members. However, a recent study revealed that STEAP1 exhibits cellular ferric reductase activity by fusing to the intracellular NADPH-binding domain of STEAP4. These findings can ultimately contribute to the development of STEAP1 targeted therapy. In contrast, the pathological functions of STEAP1 in cancer still need further investigation.

Specifications

Catalog Number	KC-4765
Cell Line Name	293T-cyno-STEAP1-Cell-Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous cyno-STEAP1 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 + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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

293T-cyno-STEAP1-cell-line was generated using a lentiviral vector expressing the cyno-STEAP1 sequence.

Characterization

Figure 1: Characterization of cyno-STEAP1 overexpression in the 293T-cyno-STEAP1 stable clone using FACS.

Figure 2: Characterization of cyno-STEAP1 in the 293T-cyno-STEAP1 stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/mL puromycin)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

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

References

1.Bhatia, V., Kamat, N.V., Pariva, T.E. et al. Targeting advanced prostate cancer with STEAP1 chimeric antigen receptor T cell and tumor-localized IL-12 immunotherapy. Nat Commun 14, 2041 (2023).
2.Nakamura H, Arihara Y, Takada K. Targeting STEAP1 as an anticancer strategy. Front Oncol. 2023 Oct 16;13:1285661. doi: 10.3389/fonc.2023.1285661. PMID: 37909017; PMCID: PMC10613890.
3.Xu M, Evans L, Bizzaro CL, Quaglia F, Verrillo CE, Li L, Stieglmaier J, Schiewer MJ, Languino LR, Kelly WK. STEAP1-4 (Six-Transmembrane Epithelial Antigen of the Prostate 1-4) and Their Clinical Implications for Prostate Cancer. Cancers (Basel). 2022 Aug 20;14(16):4034. doi: 10.3390/cancers14164034. PMID: 36011027; PMCID: PMC9406800.