TEK, also known as CD202b and 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.
Cell Line Name:
HEK293T TEK High Cell Line
Host Cell Line:
Human HEK293T cell line
One vial of frozen cells (5X106 per vial)
Stable in culture over a minimum of 10 passages
Drug screening and biological assays
70% DMEM+20% FBS+10% DMSO
Split saturated culture 1:3-1:6 every 3 days; seed out at about 1-3 × 105 cells/mL
37 °C with 5% CO2
Liquid nitrogen immediately upon receiving
Approximately 30 hours
Cell Line Generation
293T human TEK High cell line was generated using a lentiviral vector expressing the human TEK sequence.
Characterization using PCR sequencing and WB
1. Prewarm culture medium (DMEM + 10% FBS + 1μg/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 3 days; seed out at about 1-3 × 105 cells/mL.
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
1. Partanen J, Armstrong E, Mäkelä 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): 1698–707.
2. 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): 1583–7.
3. "Entrez Gene: TEK TEK tyrosine kinase, endothelial (venous malformations, multiple cutaneous and mucosal)"
4. 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.
5. 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. doi:10.1002/jsp2.1018. PMC 6686801. PMID 31463445.
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