KC-1965

HT-29-LRRC15 Cell Line

23139

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Background of HT-29-LRRC15 Cell Line

Leucine rich repeat containing 15 is a transmembrane protein frequently overexpressed in multiple tumor types as tumor antigen.

Specifications

Catalog Number	KC-1965
Cell Line Name	HT-29-LRRC15 Cell Line
Host Cell Line	HT-29
Description	Stable HT-29 cell line expressing exogenous human LRRC15 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 1μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:5 every 2-3 days; seed out at about 1-2 × 10⁵ cells/ml
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	Yes

Cell Line Generation

HT-29-LRRC15 Cell Line was generated using a lentiviral vector expressing the LRRC15 sequence.

Characterization

Figure 1: Characterization of LRRC15 overexpression in the HT-29-LRRC15 stable clone using FACS. (Antibody: Samrotamab, Kyinno, Cat#KB-1253)

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 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 1:3-1:5 every 2-3 days; seed out at about 1-2 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 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.O’Prey, J., Wilkinson, S. & Ryan, K. M. Tumor Antigen LRRC15 Impedes Adenoviral Infection: Implications for Virus-Based Cancer Therapy. J. Virol. 82, 5933–5939 (2008).
2. Wang, Y. et al. LRRC15 promotes osteogenic differentiation of mesenchymal stem cells by modulating p65 cytoplasmic/nuclear translocation. Stem Cell Res. Ther. 9, 65 (2018).