KC-1018

293T-ROR1-Cell-Line

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Background of 293T-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-1018
Cell Line Name	293T-ROR1-Cell-Line
Host Cell Line	293T
Description	Stable 293T 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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 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	NA

Cell Line Generation

293T-ROR1 cell line was generated using a lentiviral vector expressing the human ROR1 sequence.

Characterization

Figure 1

Characterization of ROR1 overexpression in the 293T ROR1 stable clone using FACS.Hybridoma or ligand binding screening with FACS.

Cell Resuscitation

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

Cell Freezing

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

References

Masiakowski P, Carroll RD (Dec 1992). A novel family of cell surface receptors with tyrosine kinase-like domain. The Journal of Biological Chemistry. 267 (36): 26181–90.2.
Reddy UR, Phatak S, Allen C, Nycum LM, Sulman EP, White PS, Biegel JA (Apr 1997). Localization of the humanRor1 gene (NTRKR1) to chromosome 1p31-p32 by fluorescence in situ hybridization and somatic cell hybridanalysis. Genomics. 41 (2): 283–5.3.
Baskar S, Kwong KY, Hofer T, Levy JM, Kennedy MG, Lee E, Staudt LM, Wilson WH, Wiestner A, Rader C (Jan2008). Unique cell surface expression of receptor tyrosine kinase ROR1 in human B-cell chronic lymphocyticleukemia. Clinical Cancer Research. 14(2): 396–404.