KC-2181

NIH/3T3-mouse-FAP Cell Line

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Background of NIH/3T3-mouse-FAP Cell Line

Fibroblast activation protein alpha (FAP) also named as prolyl endopeptidase FAP is a membrane-bound gelatinase, the expression of which is very low human tissue under physiological condition, and is high inactivated stromal fibroblasts of many human carcinoma.

Specifications

Catalog Number	KC-2181
Cell Line Name	NIH/3T3-mouse-FAP Cell Line
Host Cell Line	NIH/3T3
Description	Stable NIH/3T3 cell line expressing exogenous mouse FAP 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 + 1ug/ml Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 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 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

NIH/3T3-mouse-FAP cell line was generated using lentiviral vector expressing mouse FAP sequence.

Characterization

Figure 1: Characterization of mouse FAP overexpression in the NIH/3T3-mouse-FAP stable clone using FACS

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 1ug/ml Puromycin)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1. Rettig WJ, Su SL, Fortunato SR, Scanlan MJ, Raj BK, Garin-Chesa P, Healey JH, Old LJ (August 1994). Fibroblast activation protein: purification, epitope mapping and induction by growth factors. International Journal of Cancer. 58 (3): 385–92
2. Garin-Chesa P, Old LJ, Rettig WJ (September 1990). Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers. Proceedings of the National Academy of Sciences of the United States of America. 87 (18): 7235–9.