KC-5516

HPB-ALL-CD3E-CD3D-KO Cell Line

56421

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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	One vial 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

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.