KC-2312

293T-TEK-Middle-Cell-Line

23810

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Background of 293T-TEK-Middle-Cell-Line

TEK, also known as CD202b or Tie2, is an angiopoietin receptor that belongs to the protein tyrosine kinase family, which is expressed by endothelial cells and their progenitors, quiescent hematopoietic stem cells (HSCs), and a subpopulation of monocytes. Mutations in this gene are associated with inherited venous malformations of the skin and mucous membranes. Angiopoietin-1 (Ang-1) is an activator of CD202b to promote, maintain, and stabilize mature vessels and to maintain HSCs in quiescent state. Ang-2 is another ligand of CD202b, which is involved in postnatal angiogenesis and in antagonizing the effects of Ang-1.

Specifications

Catalog Number	KC-2312
Cell Line Name	293T-TEK-Middle-Cell-Line
Host Cell Line	Human HEK293T cell line
Description	Stable 293T cell line expressing exogenous human TEK 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+1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 every 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 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T human TEK Middle cell line was generated using a lentiviral vector expressing the human TEK sequence.

Characterization

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 1μg/mL Puromycin) in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:3-1:6 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage

References

Partanen J, Armstrong E, M?kel? TP, Korhonen J, Sandberg M, Renkonen R, Knuutila S, Huebner K, Alitalo K (April 1992). "A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains". Molecular and Cellular Biology. 12 (4): 1698ÿ707.
Boon LM, Mulliken JB, Vikkula M, Watkins H, Seidman J, Olsen BR, Warman ML (September 1994). "Assignment of a locus for dominantly inherited venous malformations to chromosome 9p". Human Molecular Genetics. 3 (9): 1583ÿ7.
"Entrez Gene: TEK TEK tyrosine kinase, endothelial (venous malformations, multiple cutaneous and mucosal)"
Sakai D, Nakamura Y, Nakai T, Mishima T, Kato S, Grad S, et al. (2012). "Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc". Nature Communications. 3: 1264. Bibcode:2012NatCo...3.1264S.
Sakai D, Schol J, Bach FC, Tekari A, Sagawa N, Nakamura Y, et al. (June 2018). "Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species". JOR Spine. 1 (2): e1018.