KC-6009

293T-mem-KLK3-Low Cell Line

61019

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Background of 293T-mem-KLK3-Low Cell Line

Kallikrein-related peptidase 3 (KLK3), also known as prostate-specific antigen (PSA), is the most useful biomarker for prostate cancer (PCa). KLK3 (Kallikrein Related Peptidase 3) is a Protein Coding gene. Early studies suggest that KLK3 is able to inhibit angiogenic processes, which is most likely dependent on its proteolytic activity. KLK3 is an important component in human semen. This protein is secreted by the epithelial cells of the prostate and its main function is to promote sperm liquefaction by decomposing seminal vesicle coagulation protein. It plays a crucial role in the reproductive process. The expression of KLK3 is strictly regulated by androgens and is commonly used as a core biomarker for prostate cancer screening in clinical practice. Diseases associated with KLK3 include Prostate Disease and Prostatitis.

Specifications

Catalog Number	KC-6009
Cell Line Name	293T-mem-KLK3-Low Cell Line
NCBI/UniProt Accession Number	NM_001648.2
Clone Number	1#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous mem KLK3 gene in low level
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 1:4-1:5 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-mem-KLK3-Low cell line was generated using a lentiviral vector expressing the mem KLK3 sequence.

Characterization

Figure 1: Characterization of mem KLK3 overexpression in the 293T-mem-KLK3-Low stable clone using FACS(Cat# KB-1832, Kyinno).

Figure 2: Characterization of mem KLK3 in the 293T stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 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 1:4-1:5 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. Koistinen H, Künnapuu J, Jeltsch M. KLK3 in the Regulation of Angiogenesis-Tumorigenic or Not? Int J Mol Sci. 2021 Dec 17;22(24):13545. doi: 10.3390/ijms222413545. PMID: 34948344; PMCID: PMC8704207.
2. Jha SK, Rauniyar K, Chronowska E, Mattonet K, Maina EW, Koistinen H, Stenman UH, Alitalo K, Jeltsch M. KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D. Elife. 2019 May 17;8:e44478. doi: 10.7554/eLife.44478. PMID: 31099754; PMCID: PMC6588350.
3. Li H, Fei X, Shen Y, Wu Z. Association of gene polymorphisms of KLK3 and prostate cancer: A meta-analysis. Adv Clin Exp Med. 2020 Aug;29(8):1001-1009. doi: 10.17219/acem/121521. PMID: 32869960.