KC-5516

HPB-ALL-CD3E-CD3D-KO Cell Line

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Background of HPB-ALL-CD3E-CD3D-KO Cell Line

The protein encoded by CD3D 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). The protein encoded by CD3E 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.

Specifications

Catalog Number	KC-5516
Cell Line Name	HPB-ALL-CD3E-CD3D-KO Cell Line
Clone Number	1B4
Host Cell Line	HPB-ALL-CD3E-KO
Description	Stable HPB-ALL-CD3E-KO clone with CD3D gene knockout, No.1B4
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	RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS
Selection Marker	NA
Morphology	Lymphoblast
Subculture	Split saturated culture 1:3-1:6 every 1-3 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 50 hours
Mycoplasma Status	Negative

Cell Line Generation

HPB-ALL-CD3E-CD3D-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of HPB-ALL-CD3E-KO Cell Line stable clone using FACS.

Figure 2: Characterization of HPB-ALL-CD3E-KO Cell Line stable clone using PCR sequencing.

Figure 3: Characterization of HPB-ALL-CD3E-KO cell line stable clone using RT-PCR sequencing.

Figure 4: Characterization of HPB-ALL-CD3E-CD3D-KO Cell Line stable clone using PCR sequencing.

Figure 5: Characterization of HPB-ALL-CD3E-CD3D-KO Cell Line stable clone using RT-PCR sequencing.

Figure 6: Characterization of HPB-ALL-CD3E-CD3D-KO Cell Line stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS) 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 1:3-1:6 every 1-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 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

https://www.ncbi.nlm.nih.gov/gene/915
https://www.ncbi.nlm.nih.gov/gene/916