KC-5223

293T-ITGB4-ITGA6 Cell Line

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Home » 293T-ITGB4-ITGA6 Cell Line

Background of 293T-ITGB4-ITGA6 Cell Line

ITGA6 encodes a member of the integrin alpha chain family of proteins. Integrins are heterodimeric integral membrane proteins composed of an alpha chain and a beta chain that function in cell surface adhesion and signaling. The encoded preproprotein is proteolytically processed to generate light and heavy chains that comprise the alpha 6 subunit. This subunit may associate with a beta 1 or beta 4 subunit to form an integrin that interacts with extracellular matrix proteins including members of the laminin family. The alpha 6 beta 4 integrin may promote tumorigenesis, while the alpha 6 beta 1 integrin may negatively regulate erbB2/HER2 signaling. Alternative splicing results in multiple transcript variants.
Integrins are heterodimers comprised of alpha and beta subunits, that are noncovalently associated transmembrane glycoprotein receptors. Different combinations of alpha and beta polypeptides form complexes that vary in their ligand-binding specificities. Integrins mediate cell-matrix or cell-cell adhesion, and transduced signals that regulate gene expression and cell growth. This gene encodes the integrin beta 4 subunit, a receptor for the laminins. This subunit tends to associate with alpha 6 subunit and is likely to play a pivotal role in the biology of invasive carcinoma. Mutations in this gene are associated with epidermolysis bullosa with pyloric atresia. Multiple alternatively spliced transcript variants encoding distinct isoforms have been found for this gene.

Specifications

Catalog NumberKC-5223
Cell Line Name293T-ITGB4-ITGA6 Cell Line
Clone Number3#
Host Cell Line293T
DescriptionStable 293T cell line expressing exogenous ITGB4 and ITGA6 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% DMEM + 20% FBS + 10% DMSO
Propagation MediumDMEM + 10% FBS + 150ug/ml Hygromycin B + 1ug/ml Puromycin
Selection MarkerPuromycin
MorphologyEpithelial
SubcultureSplit saturated culture 1:4-1:5 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

293T ITGB4 ITGA6 cell line was generated using lentiviral vector expressing ITGB4 and ITGA6 sequence.

Characterization

Figure 1: Characterization of ITGB4 and ITGA6 overexpression in the 293T ITGB4 ITGA6 stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 150ug/ml Hygromycin B + 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.Sung JS, Kang CW, Kang S, Jang Y, Chae YC, Kim BG, Cho NH. ITGB4-mediated metabolic reprogramming of cancer-associated fibroblasts. Oncogene. 2020 Jan;39(3):664-676.
2. Landowski TH, Gard J, Pond E, Pond GD, Nagle RB, Geffre CP, Cress AE. Targeting integrin α6 stimulates curative-type bone metastasis lesions in a xenograft model. Mol Cancer Ther. 2014 Jun;13(6):1558-66.
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