KC-6414

MC38-FAP-CLDN18.2-Luc2 Cell Line

62260

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Background of MC38-FAP-CLDN18.2-Luc2 Cell Line

Fibroblast activation protein-α (FAP) is a type II integral serine protease that is specifically expressed by activated fibroblasts. FAP-α displays both exopeptidase and endopeptidase/gelatinase/collagenase activities. FAP-α protein and/or activity have been associated with fibrosis, inflammation and cancer, but the protein is undetectable in most normal tissues. Cancer-associated fibroblasts (CAFs) in the tumor stroma have an abundant and stable expression of FAP, which plays an important role in promoting tumor growth, invasion, metastasis, and immunosuppression. CAF overexpression of FAP promotes tumor development and metastasis by influencing extracellular matrix remodeling, intracellular signaling, angiogenesis, epithelial-to-mesenchymal transition, and immunosuppression.
The claudin18.2 (CLDN18.2) protein, an isoform of claudin18, a member of the tight junction protein family, is a highly selective biomarker with limited expression in normal tissues and often abnormal expression during the occurrence and development of various primary malignant tumors, such as gastric cancer/gastroesophageal junction (GC/GEJ) cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer and non-small-cell lung cancer, it participates in the proliferation, differentiation and migration of tumor cells. Claudin-18.2(CLDN18.2), a transmembrane protein, is a major component of tight junctions and plays an important role in the maintenance of barrier function. Its characteristic widespread expression in tumour tissues and its exposed extracellular loops make it an ideal target for researchers to develop targeted strategies and immunotherapies for cancers of the GI tract.

Specifications

Catalog Number	KC-6414
Cell Line Name	MC38-FAP-CLDN18.2-Luc2 Cell Line
NCBI/UniProt Accession Number	NM_004460 \| NM_001002026
Clone Number	16#
Host Cell Line	Mouse MC38 Cell Line
Description	Stable MC38 cell line expressing exogenous human FAP and CLDN18.2 and luciferase 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 + 5μg/mL Puromycin + 150μg/mL Hygromycin B
Selection Marker	Puromycin \| Hygromycin B
Morphology	Fibroblast
Subculture	Split saturated culture 1:4-1:8 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 24 hours
Mycoplasma Status	Negative

Cell Line Generation

MC38-FAP-CLDN18.2-Luc2 cell line was generated using a lentiviral vector expressing the human FAP and CLDN18.2 and luciferase sequence.

Characterization

Figure 1: Characterization of human FAP and CLDN18.2 overexpression MC38 stable clone using FACS.

Figure 2: Characterization of the MC38-FAP-CLDN18.2-Luc2 cell line stable clone using Bright-Lite Luciferase Assay System in the conditions of different cell number.

Figure 3: Characterization of MC38-FAP-CLDN18.2-Luc2 cell line stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 5μg/mL Puromycin + 150μg/mL Hygromycin B) 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 1:4-1:8 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. Cao W, Xing H, Li Y, Tian W, Song Y, Jiang Z, Yu J. Claudin18.2 is a novel molecular biomarker for tumor-targeted immunotherapy. Biomark Res. 2022 May 31;10(1):38. doi: 10.1186/s40364-022-00385-1. PMID: 35642043; PMCID: PMC9153115.
4. Cha JH, Chan LC, Li CW, Hsu JL, Hung MC. Mechanisms Controlling PD-L1 Expression in Cancer. Mol Cell. 2019 Nov 7;76(3):359-370. doi: 10.1016/j.molcel.2019.09.030. Epub 2019 Oct 24. PMID: 31668929; PMCID: PMC6981282.