KC-6420

293T-FASL Cell Line

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Background of 293T-FASL Cell Line

FASLG belongs to the tumor necrosis factor (TNF) family and is encoded by a gene located on chromosome 1q23 in human (NCBI Gene ID:356); it is normally stored in the cytoplasm in secretory vesicles and can be expressed as two different isoforms: a 37 kDa membrane-bound protein (mFASLG) and a 26–29 kDa soluble variant (sFASLG), produced by a metalloproteinase-mediated cleavage of the mFASLG. The FASLG gene is composed of four exons and is located on chromosome 1 both in human and mice. It encodes a type II protein, meaning that the N-terminal domain is intracellular.

Specifications

Catalog Number	KC-6420
Cell Line Name	293T-FASL Cell Line
NCBI/UniProt Accession Number	NM_000639
Clone Number	8#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous FASL 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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial-like
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 28 hours
Mycoplasma Status	Negative

Cell Line Generation

293T-FASL cell line was generated using a lentiviral vector expressing the human FASL sequence.

Characterization

Figure 1: Characterization of FASL overexpression in the 293T-FASL stable clone using PCR sequencing.

Figure 2: Characterization of FASL overexpression in the 293T-FASL stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM+10% FBS+1μg/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% DMEM + 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.