KC-5455

NIH/3T3-FAP-Cell-Line

56261

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

FAP, also known as FAPalpha, is a type II transmembrane glycoprotein that belongs to the serine protease family, which is selectively expressed in reactive stromal fibroblasts of epithelial cancers, granulation tissue of healing wounds, and malignant cells of bone and soft tissue sarcomas. FAP is thought to be involved in the control of fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair, and epithelial carcinogenesis. Acts as a tumor suppressor in melanocytic cells through regulation of cell proliferation and survival in a serine protease activity-independent manner.

Specifications

Catalog Number	KC-5455
Cell Line Name	NIH/3T3-FAP-Cell-Line
Clone Number	4#
Host Cell Line	NIH/3T3
Description	Stable NIH/3T3 clone expressing exogenous 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	DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 2μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Fibroblast
Subculture	Split saturated culture 1:6-1:8 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 22 hours
Mycoplasma Status	Negative

Cell Line Generation

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

Characterization

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

Figure 2: Characterization of FAP in the NIH/3T3-FAP stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 2μg/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 1:6-1:8 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. Puré E, Blomberg R (August 2018). Pro-tumorigenic roles of fibroblast activation protein in cancer: back to the basics. Oncogene. 37 (32): 4343–4357. doi:10.1038/s41388-018-0275-3. PMC 6092565. PMID 29720723.
2.Xin L, Gao J, Zheng Z, Chen Y, Lv S, Zhao Z, Yu C, Yang X, Zhang R. Fibroblast Activation Protein-α as a Target in the Bench-to-Bedside Diagnosis and Treatment of Tumors: A Narrative Review. Front Oncol. 2021 Aug 19;11:648187. doi: 10.3389/fonc.2021.648187. PMID: 34490078; PMCID: PMC8416977.
3. Busek P, Mateu R, Zubal M, Kotackova L, Sedo A (June 2018). Targeting fibroblast activation protein in cancer - Prospects and caveats. Frontiers in Bioscience. 23: 1933–1968. doi:10.2741/4682. PMID 29772538.
4.Niedermeyer J, Garin-Chesa P, Kriz M, Hilberg F, Mueller E, Bamberger U, Rettig WJ, Schnapp A (April 2001). Expression of the fibroblast activation protein during mouse embryo development. The International Journal of Developmental Biology. 45 (2): 445–7. PMID 11330865.