KC-3866

3A9 CD3ED Cell Line

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Background of 3A9 CD3ED Cell Line

The CD3 antigen is an important marker on the surface of T cells, and it forms the TCR/CD3 receptor complex with the T-cell receptor (TCR) through non-covalent bonds. The CD3 complex includes four protein chains: CD3δ, CD3ε, CD3γ, and CD3ζ. Among these, the CD3δ/CD3ε and CD3γ/CD3ε form heterodimers with the α/β chains of the TCR to create the TCR-CD3 complex.After stimulation by antigens, conformational changes occur in the cytoplasmic tail regions of the CD3 complex. Proteins such as LCK (lymphocyte-specific protein tyrosine kinase) and Src family protein tyrosine kinases phosphorylate the tyrosine residues within the ITAMs (immunoreceptor tyrosine-based activation motifs) of the CD3 complex, creating binding sites for proteins with SH2 domains. Additionally, ZAP70 and the linker for activation of T cells (LAT) are phosphorylated, leading to the activation of downstream pathways, such as MAPK (mitogen-activated protein kinase) and NF-κB signaling cascades. Under the influence of multiple transcription factors, this can result in T cell proliferation, migration, cytokine production, and effector functions.

Specifications

Catalog Number	KC-3866
Cell Line Name	3A9 CD3ED Cell Line
Host Cell Line	3A9
Description	Stable 3A9 T cell hybridoma clone expressing exogenous human CD3E and CD3D gene
Quantity	One vial of frozen cells (≥2×10⁶ cells)
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 + 1µg/mL Puromycin
Selection Marker	Puromycin
Morphology	lymphoblast-like
Subculture	Split the saturated culture at a ratio of 1:4~1:6 every 2~3 days; seed out at about 1-3 x 10^{5 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

3A9-CD3ED cell line was generated using a lentiviral vector expressing the human CD3E and CD3D sequence.

Characterization

Figure 1: Characterization of human CD3E in 3A9 using FACS.

Figure 2: Characterization of human CD3E overexpression in 3A9-CD3ED stable clone using FACS.

Figure 3: Characterization of 3A9-CD3ED Cell Line stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (RPMI1640 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~ 1:6 every 2~3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

LiverTox: Clinical and Research Information on Drug-Induced Liver Injury [Internet]. Bethesda (MD): National Institute of Diabetes and Digestive and Kidney Diseases; 2012–. Muromonab-CD3. 2020 Dec 28. PMID: 31643905.
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.
Borroto A, Abia D, Alarcón B. Crammed signaling motifs in the T-cell receptor. Immunol Lett. 2014 Sep;161(1):113-7. doi: 10.1016/j.imlet.2014.05.007. Epub 2014 May 27. PMID: 24877875.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.