KC-4176

Jurkat-NFAT-Luc2-CD3E-KO-1B3-Cell-Line

42390

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Background of Jurkat-NFAT-Luc2-CD3E-KO-1B3-Cell-Line

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.

Specifications

Catalog Number	KC-4176
Cell Line Name	Jurkat-NFAT-Luc2-CD3E-KO-1B3-Cell-Line
Host Cell Line	Jurkat-NFAT-Luc2-reporter
Description	Stable Jurkat-NFAT-Luc2 clone with human CD3E gene knockout, No.1B3
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+300 μg/mL Hygromycin B
Selection Marker	N/A
Morphology	Lymphoblast
Subculture	Split saturated culture 1:4-1:6 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 26 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

Characterization

Figure 1: Characterization of CD3E knockout in Jurkat-NFAT-Luc2 using PCR sequencing.

Figure 2: Characterization of CD3E knockout in Jurkat-NFAT-Luc2 using RT-PCR sequencing.

Figure 3: Characterization of CD3E knockout in Jurkat-NFAT-Luc2 using FACS.

Figure 4: Chareacterization of Jurkat-NFAT-Luc2-CD3-KO clones and Jurkat-NFAT-Luc2 parental cells treated by 20μg/mL OKT3 for 6 hours using Bright-Glo system.

Figure 5. Chareacterization of Jurkat-NFAT-Luc2-CD3-KO clones and Jurkat-NFAT-Luc2 cell line were seeded into the 96-well plate, and treated with OKT3 at a maximum concentration of 20μg/mL for 16 hours, then readout with Bright-Glo system.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS+300 μg/mL Hygromycin B)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 1:4-1:6 every 2-3 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/916