KC-6496

CHOK1-FAS Cell Line

62482

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Background of CHOK1-FAS Cell Line

FAS (TNFRSF6) is the sixth member of the Tumor Necrosis Factor Receptor (TNFR) superfamily (NCBI gene ID:355). It is a membrane-bound trimeric protein playing a key role in apoptosis. The FAS gene, located on the long-arm of the chromosome 10 in human and on the chromosome 19 in mice, is composed of 9 exons encoding a type-I protein. Exons 1 to 5, 6 and 7 to 9 encode the extracellular domain (ECD), the transmembrane domain (TD) and the intracellular domain (ICD) respectively. A functional domain called the Death Domain (DD), entirely encoded by the exon 9, defines the subfamily of Death Receptors such as the TNF-Receptor 1 (TNFRSF1A), DR3 (TNFRSF25), FAS (TNFSFR6), and the TRAIL receptors TRAILR1 and TRAILR2 (TNFRSF10-A and -B respectively). The mature FAS protein has 319 amino acids and a predicted molecular weight of 48 kD. FAS is expressed as a trimer mainly on activated lymphocytes and virtually in all tissues. Upon interaction with its cognate ligand FASLG, FAS triggers a biochemical cascade leading to the organized cell destruction.

Specifications

Catalog Number	KC-6496
Cell Line Name	CHOK1-FAS Cell Line
NCBI/UniProt Accession Number	NM_000043.3
Clone Number	9#
Host Cell Line	CHOK1
Description	Stable CHOK1 clone expressing exogenous FAS gene
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	70% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 10μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Fibroblastoid cells growing as a monolayer
Subculture	Split the saturated culture at a ratio of 1:4-1:5 every 2-3 days; seed out at about 1-3 x 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

CHOK1-FAS cell line was generated using a lentiviral vector expressing the FAS sequence.

Characterization

Figure 1:Characterization of FAS overexpression in the CHOK1-FAS stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640+10% FBS+10μg/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 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Magerus A, Bercher-Brayer C, Rieux-Laucat F. The genetic landscape of the FAS pathway deficiencies. Biomed J. 2021 Aug;44(4):388-399. doi: 10.1016/j.bj.2021.06.005. Epub 2021 Jun 24. PMID: 34171534; PMCID: PMC8514852.