KC-5425

293T-LRP6-D1/2 Cell Line

56834

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Background of 293T-LRP6-D1/2 Cell Line

LRP6 is a member of the low-density lipoprotein receptor superfamily of cell-surface receptors. It is required for the activation of the Wnt/β-catenin signalling pathway. LRP6 is detected in different tissue types and is involved in numerous biological activities such as cell proliferation, specification, metastatic cancer, and embryonic development. In human, LRP6 overexpression and mutations have been reported in multiple complex diseases including hypertension, atherosclerosis, and cancers.

Specifications

Catalog Number	KC-5425
Cell Line Name	293T-LRP6-D1/2 Cell Line
Clone Number	7-13#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous human LRP6 D1/2 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-LRP6-D1/2 cell line was generated using a lentiviral vector expressing the LRP6 D1/2 sequence.

Characterization

Figure 1: Characterization of LRP6 D1/2 overexpression in the 293T LRP6 D1/2 stable clone using FACS(Kyinno, Cat#KB-1433).

Figure 2: Characterization of LRP6 D1/2 and its mutants overexpressing in 293T stable clones 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. Alrefaei AF, Abu-Elmagd M. LRP6 Receptor Plays Essential Functions in Development and Human Diseases. Genes (Basel). 2022 Jan 10;13(1):120. doi: 10.3390/genes13010120. PMID: 35052459; PMCID: PMC8775365.
2. Xue W, Zhu B, Zhao K, Huang Q, Luo H, Shou Y, Huang Z, Guo H. Targeting LRP6: A new strategy for cancer therapy. Pharmacol Res. 2024 Jun;204:107200. doi: 10.1016/j.phrs.2024.107200. Epub 2024 May 6. PMID: 38710241.
3. Li L, Zhao L, Yang J, Zhou L. Multifaceted effects of LRP6 in cancer: exploring tumor development, immune modulation and targeted therapies. Med Oncol. 2024 Jun 19;41(7):180. doi: 10.1007/s12032-024-02399-1. PMID: 38898247.