KC-4467

293T-mouse-CD4-Cell-Line

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Background of 293T-mouse-CD4-Cell-Line

The CD4 gene encodes the CD4 membrane glycoprotein of T lymphocytes. CD4 antigen acts as a co-receptor with T cell receptors on T lymphocytes to recognize antigens displayed by antigen-presenting cells in the context of Class II MHC molecules. CD4 antigen is also a major receptor that enters the human immunodeficiency virus through interaction with the HIV Env gp120 subunit. The gene is expressed not only in T lymphocytes, but also in B cells, macrophages, granulocytes, and in various regions of the brain.

Specifications

Catalog Number	KC-4467
Cell Line Name	293T-mouse-CD4-Cell-Line
Host Cell Line	293T
Description	Stable HEK293T clone expressing exogenous mouse CD4 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 1:4-1:8 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

Cell Line Generation

293T mouse CD4 cell line was generated using a lentiviral vector expressing the mouse CD4 sequence.

Characterization

Figure 1: Characterization of mouse CD4 in the 293T mouse CD4 stable clone using PCR sequencing.

Figure 2: Characterization of mouse CD4 overexpression in the 293T mouse CD4 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

Ruterbusch M, Pruner KB, Shehata L, Pepper M. In Vivo CD4⁺ T Cell
Differentiation and Function: Revisiting the Th1/Th2 Paradigm. Annu Rev Immunol.
2020 Apr 26;38:705-725. doi: 10.1146/annurev-immunol-103019-085803. PMID:
Dimeric arrangement of the parathyroid hormone receptor and a structural mechanism for ligand-induced dissociation. PMID: 20172855 PMCID: PMC2852981 DOI: 10.1074/jbc.M109.093138.
Tang H, Jiang X, Zhang J, Pei C, Zhao X, Li L, Kong X. Teleost
CD4⁺ helper T cells: Molecular characteristics and functions and
comparison with mammalian counterparts. Vet Immunol Immunopathol. 2021
Oct;240:110316. doi: 10.1016/j.vetimm.2021.110316. Epub 2021 Aug 25. PMID:
Lange T, Born J, Westermann J. Sleep Matters: CD4⁺ T Cell Memory
Formation and the Central Nervous System. Trends Immunol. 2019
Aug;40(8):674-686. doi: 10.1016/j.it.2019.06.003. Epub 2019 Jun 28. PMID: