KC-2269

293T-cyno-CD3E-Cell-Line

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Background of 293T-cyno-CD3E-Cell-Line

Part of the TCR-CD3 complex present on T-lymphocyte cell surface that plays an essential role in adaptive immune response. When antigen presenting cells (APCs) activate T-cell receptor (TCR), TCR-mediated signals are transmitted across the cell membrane by the CD3 chains CD3D, CD3E, CD3G and CD3Z. All CD3 chains contain immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic domain. Upon TCR engagement, these motifs become phosphorylated by Src family protein tyrosine kinases LCK and FYN, resulting in the activation of downstream signaling pathways. In addition of this role of signal transduction in T-cell activation, CD3E plays an essential role in correct T-cell development. Initiates the TCR-CD3 complex assembly by forming the two heterodimers CD3D/CD3E and CD3G/CD3E. Participates also in internalization and cell surface down-regulation of TCR-CD3 complexes via endocytosis sequences present in CD3E cytosolic region.

Specifications

Catalog Number	KC-2269
Cell Line Name	293T-cyno-CD3E-Cell-Line
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous cyno CD3E 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 cyno CD3E cell line was generated using a lentiviral vector expressing the cyno CD3E sequence.

Characterization

Figure 1: Characterization of cyno CD3E overexpression in the 293T cyno CD3E stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 1 μg/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

Fischer A, de Saint Basile G, Le Deist F. CD3 deficiencies. Curr Opin Allergy Clin Immunol. 2005 Dec;5(6):491-5. doi: 10.1097/01.all.0000191886.12645.79. PMID: 16264327.
Vanacker H, Vinceneux A, Nicolas-Virelizier E, Brahmi M, Cassier PA. Anticorps bispécifiques ciblant CD3 en oncologie solide et onco-hématologie [Bispecific antibodies targeting CD3 in oncology and hematology]. Bull Cancer. 2021 Oct;108(10S):S181-S194. French. doi: 10.1016/j.bulcan.2021.06.003. PMID: 34920802.