KC-5946

MC38-FGFR2b-GFP-Luc2-Cell-Line

60387

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Background of MC38-FGFR2b-GFP-Luc2-Cell-Line

Fibroblast growth factor receptor 2 (FGFR2) also known as CD332 is a receptor for fibroblast growth factor, it plays an important role in embryonic development and tissue repair, especially bone and blood vessels after binding with its ligands. FGFR2 was also found as a cancer diver gene in numerous cancers due to amino acid or fusion mutation.
Luciferase is an oxidative enzyme that can produce bioluminescence with addition of luciferin, but doesn’t need an external light source unlike fluorescent proteins. Photo emission can be detected directly by light sensitive devices. Such as luminometers or modified microscopes. Luciferase is widely used in many fields of biological research, such as transcriptional activity, kinase or other enzyme activity, cellular ATP level, and whole animal imaging.
Green fluorescent protein (GFP) is a β - barrel protein 1 composed of 238 amino acids with a molecular weight of approximately 27 kDa. GFP was isolated from the crystal jellyfish Aequorea Victoria. GFP can convert the blue fluorescence emitted by jellyfish luminescent protein through chemical reactions into green fluorescence through energy transfer. The excitation wavelength of GFP is 488 nm, and there is an emission peak at approximately 507 nm.

Specifications

Catalog Number	KC-5946
Cell Line Name	MC38-FGFR2b-GFP-Luc2-Cell-Line
NCBI/UniProt Accession Number	NM_022970.3
Clone Number	2#
Host Cell Line	MC38
Description	Stable MC38 clone expressing exogenous FGFR2b and GFP 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	70% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS
Selection Marker	NA
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 20 hours
Mycoplasma Status	Negative

Cell Line Generation

MC38-FGFR2b-GFP-Luc2-cell-line was generated using a lentiviral vector expressing the FGFR2b-GFP-Luc2 sequence.

Characterization

Figure 1: Characterization of FGFR2B overexpression in the MC38-FGFR2b-GFP-Luc2 stable clone using FACS.(Primary antibody: Bemarituzumab(FGFR2b), Cat#KA-1202, Kyinno)

Figure 2: Characterization of the MC38-FGFR2b-GFP-Luc2-cell-line stable clone using Bright-Lite Luciferase Assay System in the conditions of different cell number.

Figure 3: Characterization of FGFR2b in the MC38-FGFR2b-GFP-Luc2 stable clone using PCR sequencing.

Figure 4: Characterization of GFP overexpression in the MC38-FGFR2b-GFP-Luc2 stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS)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.Hunter DJ, Kraft P, Jacobs KB, Cox DG, Yeager M, Hankinson SE, Wacholder S, Wang Z, Welch R, Hutchinson A, Wang J, Yu K, Chatterjee N, Orr N, Willett WC, Colditz GA, Ziegler RG, Berg CD, Buys SS, McCarty CA, Feigelson HS, Calle EE, Thun MJ, Hayes RB, Tucker M, Gerhard DS, Fraumeni JF, Hoover RN, Thomas G, Chanock SJ (Jul 2007). "A genome-wide association study identifies alleles in FGFR2 associated with risk of sporadic postmenopausal breast cancer". Nature Genetics.
2.Greer LF, Szalay AA (2002). "Imaging of light emission from the expression of luciferases in living cells and organisms: a review". Luminescence. 17 (1): 43–74. doi:10.1002/bio.676. PMID 11816060.
3.Pieribone V, Gruber D. Aglow in the Dark: The Revolutionary Science of Biofluorescence. Cambridge: Belknap Press. 2006. ISBN 0-674-01921-0. OCLC 60321612. Popular science book describing history and discovery of GFP