KC-2310

CHOK1-cyno-CD3ED Cell Line

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Home » CHOK1-cyno-CD3ED Cell Line

Background of CHOK1-cyno-CD3ED Cell Line

CD3E, the protein encoded by this gene is the CD3-epsilon polypeptide, which together with CD3-gamma, -delta and -zeta, and the T-cell receptor alpha/beta and gamma/delta heterodimers, forms the T-cell receptor-CD3 complex. This complex plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. The genes encoding the epsilon, gamma and delta polypeptides are located in the same cluster on chromosome 11. The epsilon polypeptide plays an essential role in T-cell development. Defects in this gene cause immunodeficiency. This gene has also been linked to a susceptibility to type I diabetes in women.
CD3D, the protein encoded by this gene is part of the T-cell receptor/CD3 complex (TCR/CD3 complex) and is involved in T-cell development and signal transduction. The encoded membrane protein represents the delta subunit of the CD3 complex, and along with four other CD3 subunits, binds either TCR alpha/beta or TCR gamma/delta to form the TCR/CD3 complex on the surface of T-cells. Defects in this gene are a cause of severe combined immunodeficiency autosomal recessive T-cell-negative/B-cell-positive/NK-cell-positive (SCIDBNK). Two transcript variants encoding different isoforms have been found for this gene. Other variants may also exist, but the full-length natures of their transcripts has yet to be defined.

Specifications

Catalog NumberKC-2310
Cell Line NameCHOK1-cyno-CD3ED Cell Line
Host Cell LineCHOK1
DescriptionStable CHOK1 cell line expressing exogenous cyno CD3ED gene
QuantityOne vial of frozen cells (≥2-106/vial)
StabilityStable in culture over a minimum of 10 passages
ApplicationDrug screening and biological assays
Freezing Medium70% RPMI1640 + 20% FBS + 10% DMSO
Propagation MediumRPMI1640 + 10% FBS + 10μg/ml Puromycin
Selection MarkerPuromycin
MorphologyEpithelial
SubcultureSplit saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 105 cells/ml
Incubation37 °C with 5% CO2
StorageLiquid nitrogen immediately upon receiving
Doubling TimeApproximately 30 hours
Mycoplasma StatusNegative
In Vivo ValidationNA

Cell Line Generation

CHOK1 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 CHOK1 cyno CD3ED stable clone using FACS.

Cell Resuscitation

  1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10μg/mL Puromycin)in a 37°C water bath.
  2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
  3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
  4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
  5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
  6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
  7. Incubate the flask at 37°C, 5% CO2 incubator.
  8. Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 105 cells/mL.

Cell Freezing

  1. Prepare the freezing medium (70% RPMI1640 + 20% FBS + 10% DMSO) fresh immediately before use.
  2. Keep the freezing medium on ice and label cryovials.
  3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
  4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
  5. Resuspend the cells at a density of at least 3×106 cells/mL in chilled freezing medium.
  6. Aliquot 1 mL of the cell suspension into each cryovial.
  7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
  8. Transfer vials to liquid nitrogen for long-term storage

References

  1. Giurisato E, McIntosh DP, Tassi M, Gamberucci A, Benedetti A. T cell receptor can be recruited to a subset of plasma membrane rafts, independently of cell signaling and attendantly to raft clustering. J Biol Chem. 2003 Feb 28;278(9):6771-8.
  2. Phillips JH, Hori T, Nagler A, Bhat N, Spits H, Lanier LL. Ontogeny of human natural killer (NK) cells: fetal NK cells mediate cytolytic function and express cytoplasmic CD3 epsilon,delta proteins. J Exp Med. 1992 Apr 1;175(4):1055-66.

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.
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