KC-2079-DW

CT26-ROR1 Cell Line

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Background of CT26-ROR1 Cell Line

ROR1, also named as NTRKR1, is a transmembrane protein belong to the receptor tyrosine kinase-like orphan receptor (ROR) family. ROR1 modulates neurite growth in the central nervous system. ROR1 has recently found to be overexpressed on cancer stem cell, which play a function role in promoting cancer cell migration, invasion or spheroid formation.

Specifications

Catalog Number	KC-2079-DW
Cell Line Name	CT26-ROR1 Cell Line
Clone Number	NA
Host Cell Line	Mouse CT26 Cell Line
Description	Stable CT26 clone expressing exogenous human ROR1 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 + 10μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Fibroblast
Subculture	Split saturated culture 1:2-1:4 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
In Vivo Validation	Yes

Cell Line Generation

CT26 ROR1 cell line was generated using a lentiviral vector expressing the human ROR1 sequence.

Characterization

Figure 1: Characterization of human ROR1 overexpression CT26 using FACS.

Figure 2: Characterization of human ROR1overexpression CT26 ROR1 stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10μg/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 1:2-1:4 every 2-3 days; seed out at about 1-3 × 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.Kipps TJ. ROR1: an orphan becomes apparent. Blood. 2022 Oct 6;140(14):1583-1591. doi: 10.1182/blood.2021014760. PMID: 35580162; PMCID: PMC10653015.
2. Osorio-Rodríguez DA, Camacho BA, Ramírez-Segura C. Anti-ROR1 CAR-T cells: Architecture and performance. Front Med (Lausanne). 2023 Feb 17;10:1121020. doi: 10.3389/fmed.2023.1121020. PMID: 36873868; PMCID: PMC9981679.
3. Liu D, Gunther K, Enriquez LA, Daniels B, O\'Mara TA, Tang K, Spurdle AB, Ford CE. ROR1 is upregulated in endometrial cancer and represents a novel therapeutic target. Sci Rep. 2020 Aug 17;10(1):13906. doi: 10.1038/s41598-020-70924-z. Erratum in: Sci Rep. 2022 Jun 28;12(1):10918. doi: 10.1038/s41598-022-15043-7. PMID: 32807831; PMCID: PMC7431863.