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	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% 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.