KC-4411

Jurkat-CD3ED-KO-cyno-CD3ED-Cell-Line

46187

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Background of Jurkat-CD3ED-KO-cyno-CD3ED-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.CD3D plays an essential role in thymocyte differentiation. Indeed, participates in correct intracellular TCR-CD3 complex assembly and surface expression. In absence of a functional TCR-CD3 complex, thymocytes are unable to differentiate properly. Interacts with CD4 and CD8 and thus serves to establish a functional link between the TCR and coreceptors CD4 and CD8, which is needed for activation and positive selection of CD4 or CD8 T-cells.

Specifications

Catalog Number	KC-4411
Cell Line Name	Jurkat-CD3ED-KO-cyno-CD3ED-Cell-Line
Host Cell Line	Jurkat
Description	Stable Jurkat-CD3ED-KO clone expressing exogenous cyno-CD3ED 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% RPMI 1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI 1640 + 10% FBS + 0.75μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Lymphoblast
Subculture	Split saturated culture 1:3-1:5 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 26 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

Jurkat-CD3ED-KO-cyno-CD3ED-cell-line was generated using a lentiviral vector expressing the cyno-CD3ED sequence.

Characterization

Figure 1: Characterization of cyno-CD3ED overexpression in the Jurkat-CD3ED-KO-cyno-CD3ED stable clone using FACS.

Figure 2: Characterization of cyno-CD3ED in the Jurkat-CD3ED-KO stable clone using FACS.

Figure 3: Characterization of CD3ED in the Jurkat stable clone using FACS.

Figure 4: Characterization of cyno-CD3ED in the Jurkat-CD3ED-KO-cyno-CD3ED stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (RPMI 1640 supplemented with 10% FBS and 0.75μg/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 1:3-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI 1640 + 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.