KC-5152

A549-FGFR1-Flag Cell Line

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Home » A549-FGFR1-Flag Cell Line

Background of A549-FGFR1-Flag Cell Line

FGFR1 is a member of the Fibroblast Growth Factor family, comprising of 4 receptors and 18 Ligands.The protein encoded by this gene is a member of the fibroblast growth factor receptor (FGFR) family, where amino acid sequence is highly conserved between members and throughout evolution. FGFR family members differ from one another in their ligand affinities and tissue distribution. A full-length representative protein consists of an extracellular region, composed of three immunoglobulin-like domains, a single hydrophobic membrane-spanning segment and a cytoplasmic tyrosine kinase domain. The extracellular portion of the protein interacts with fibroblast growth factors, setting in motion a cascade of downstream signals, ultimately influencing mitogenesis and differentiation. This particular family member binds both acidic and basic fibroblast growth factors and is involved in limb induction. Mutations in this gene have been associated with Pfeiffer syndrome, Jackson-Weiss syndrome, Antley-Bixler syndrome, osteoglophonic dysplasia, and autosomal dominant Kallmann syndrome 2. Chromosomal aberrations involving this gene are associated with stem cell myeloproliferative disorder and stem cell leukemia lymphoma syndrome. Alternatively spliced variants which encode different protein isoforms have been described; however, not all variants have been fully characterized.

Specifications

Catalog NumberKC-5152
Cell Line NameA549-FGFR1-Flag Cell Line
NCBI/UniProt Accession NumberNM_023110.3
Clone Number11-1#
Host Cell LineA549
DescriptionA549 cell line stably expressing exogenous FGFR1-Flag gene
QuantityTwo vials of frozen cells (≥2-106/vial)
StabilityStable in culture over a minimum of 10 passages
ApplicationDrug screening and biological assays
Freezing Medium70% RPMI1640 + 20% FBS + 10% DMSO
Propagation MediumRPMI1640+ 10% FBS + 1 μg/mL Puromycin
Selection MarkerPuromycin
MorphologyEpithelial
SubcultureSplit saturated culture 1:3-1:6 every 2-3 days; seed out at about 1-3 × 105 cells/mL
Incubation37 °C with 5% CO2
StorageLiquid nitrogen immediately upon receiving
Doubling TimeApproximately 30 hours
Mycoplasma StatusNegative

Cell Line Generation

A549-FGFR1-Flag cell line was generated using a lentiviral vector expressing the FGFR1-Flag sequence.

Characterization

Figure1: Characterization of FGFR1-Flag overexpressing in A549 stable clones using FACS.

Figure2: Characterization of A549-FGFR1-Flag cell line stable clone using PCR sequencing.

Cell Resuscitation

  1. Prewarm culture medium (RPMI1640+ 10% FBS + 1 μg/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 1:3-1:6 every 2-3 days; seed out at about 1-3 × 105 cells/mL.

Cell Freezing

  1. Prepare the freezing medium (70% RPMI1640 + 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. Welander J, Łysiak M, Brauckhoff M, Brunaud L, Söderkvist P, Gimm O. Activating FGFR1 Mutations in Sporadic Pheochromocytomas. World J Surg. 2018 Feb;42(2):482-489. doi: 10.1007/s00268-017-4320-0. PMID: 29159601; PMCID: PMC5762800.
  2. Gadgeel SM, Chen W, Cote ML, Bollig-Fischer A, Land S, Schwartz AG, Bepler G. Fibroblast growth factor receptor 1 amplification in non-small cell lung cancer by quantitative real-time PCR. PLoS One. 2013 Nov 8;8(11):e79820. doi: 10.1371/journal.pone.0079820. PMID: 24255716; PMCID: PMC3821849.
  3. Park S, Lee M, Cho KJ, Kim SB, Roh JL, Choi SH, Nam SY, Kim SY, Song JS. Association Between Fibroblast Growth Factor Receptor 1 Gene Amplification and Human Papillomavirus Prevalence in Tonsillar Squamous Cell Carcinoma With Clinicopathologic Analysis. J Histochem Cytochem. 2018 Jul;66(7):511-522. doi: 10.1369/0022155418761652. Epub 2018 Mar 19. PMID: 29553868; PMCID: PMC6055260.
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