KC-4454

293T-cyno-CD4-Cell-Line

46484

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

This gene encodes the CD4 membrane glycoprotein of T lymphocytes. The CD4 antigen acts as a coreceptor with the T-cell receptor on the T lymphocyte to recognize antigens displayed by an antigen presenting cell in the context of class II MHC molecules. The CD4 antigen is also a primary receptor for entry of the human immunodeficiency virus through interactions with the HIV Env gp120 subunit. This gene is expressed not only in T lymphocytes, but also in B cells, macrophages, granulocytes, as well as in various regions of the brain. The protein functions to initiate or augment the early phase of T-cell activation, and may function as an important mediator of indirect neuronal damage in infectious and immune-mediated diseases of the central nervous system. Multiple alternatively spliced transcript variants encoding different isoforms have been identified in this gene.

Specifications

Catalog Number	KC-4454
Cell Line Name	293T-cyno-CD4-Cell-Line
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous luciferase gene under the control of cyno CD4 signaling pathway
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Cell model for monitoring cyno CD4 signaling pathway
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
In Vivo Validation	NA

Cell Line Generation

293T-cyno-CD4 cell line was generated using lentivirus expressing cyno-CD4 sequence.

Characterization

Figure 1. Characterization of cyno-CD4 over-expression in the 293T-cyno-CD4 stable clone using FACS.

Figure 2. Characterization of cyno-CD4 in the 293T-cyno-CD4 stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 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

Indraccolo S, Mion M, Biagiotti R, Romagnani S, Morfini M, Longo G, Zamarchi R, Chieco-Bianchi L, Amadori A. Genetic variability of the human CD4 V2 domain. Immunogenetics. 1996;44(1):70-2. doi: 10.1007/BF02602658. PMID: 8613144.
Kim KC, Kim HG, Roh TY, Park J, Jung KM, Lee JS, Choi SY, Kim SS, Choi BS. The effect of CD4 receptor downregulation and its downstream signaling molecules on HIV-1 latency. Biochem Biophys Res Commun. 2011 Jan 14;404(2):646-51. doi: 10.1016/j.bbrc.2010.12.032. Epub 2010 Dec 10. PMID: 21146497.
Wu X, Tschumper RC, Gutierrez A Jr, Mihalcik SA, Nowakowski GS, Jelinek DF. Selective induction of DNA repair pathways in human B cells activated by CD4+ T cells. PLoS One. 2010 Dec 16;5(12):e15549. doi: 10.1371/journal.pone.0015549. PMID: 21179576; PMCID: PMC3002972..