KC-3356

293T-CRTAM-Cell-Line

34346

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Background of 293T-CRTAM-Cell-Line

CRTAM, also known as CD355, is a class I MHC restricted T cell related molecule in the immunoglobulin superfamily, highly expressed in the cerebellum, especially in Purkinje neurons.CRTAM expression is restricted to activated class-I MHC-restricted T cells, including NKT and CD8 T cells. CRTAM represents one of the most highly expressed surface markers of activated human CD8 T cells and NKT cells, suggesting it may have diagnostic uses in various human viral and autoimmune diseases.

Specifications

Catalog Number	KC-3356
Cell Line Name	293T-CRTAM-Cell-Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous CRTAM gene in high 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 1:4-1:8 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
In Vivo Validation	NA

Cell Line Generation

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

Characterization

Figure 1: Characterization of CRTAM overexpression in the 293T-CRTAM stable clone using FACS.

Figure 2: Characterization of CRTAM in the 293T-CRTAM stable clone using PCR sequencing.

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 1:4-1:8 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

Garay E, Patiño-López G, Islas S, Alarcón L, Canche-Pool E, Valle-Rios R, Medina-Contreras O, Granados G, Chávez-Munguía B, Juaristi E, Ortiz-Navarrete V, González-Mariscal L. CRTAM: A molecule involved in epithelial cell adhesion. J Cell Biochem. 2010 Sep 1;111(1):111-22. doi: 10.1002/jcb.22673. PMID: 20556794.
Patiño-Lopez G, Hevezi P, Lee J, Willhite D, Verge GM, Lechner SM, Ortiz-Navarrete V, Zlotnik A. Human class-I restricted T cell associated molecule is highly expressed in the cerebellum and is a marker for activated NKT and CD8+ T lymphocytes. J Neuroimmunol. 2006 Feb;171(1-2):145-55. doi: 10.1016/j.jneuroim.2005.09.017. Epub 2005 Nov 18. PMID: 16300832.