KC-0993

293T-CD133-Cell-Line

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Background of 293T-CD133-Cell-Line

CD133, also known as prominin-1, is a member of pentaspan transmembrane glycoproteins and expressed in hematopoietic stem cells, endothelial progenitor cells, glioblastoma, neuronal and glial stem cells, and some other cell types. CD133 is also the most used cancer stem cells maker, which might be a potential target for cancer therapy.

Specifications

Catalog Number	KC-0993
Cell Line Name	293T-CD133-Cell-Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous CD133 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 1:4-1:8 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 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T-CD133-cell-line was generated using a lentiviral vector expressing the CD133 sequence.

Characterization

Figure 1: Characterization of CD133 overexpression in the 293T-CD133 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 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 1:4-1:8 every 2-3 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

Yin AH, Miraglia S, Zanjani ED, Almeida-Porada G, Ogawa M, Leary AG, Olweus J, Kearney J, Buck DW (1997). AC133, is a novel marker for human hematopoietic stem and progenitor cells. Blood. 90 (12): 5002–5012.
Corbeil D, Fargeas CA, Huttner WB (2001). Rat prominin, like its mouse and human orthologues, is a pentaspan membrane glycoprotein. Biochem Biophys Res Commun. 285 (4): 939–44. doi:10.1006/bbrc.2001.5271.
Irollo E, Pirozzi G (2013). CD133: to be or not to be, is this the real question?. Am J Transl Res. 5 (6): 563–81.
Horn PA, Tesch H, Staib P, Kube D, Diehl V, Voliotis D (1999). Expression of AC133, a novel hematopoietic precursor antigen, on acute myeloid leukemia cells. Blood. 93 (4): 1435–37. PMID 10075457.
Corbeil D, Röper K, Hellwig A, Tavian M, Miraglia S, Watt SM, Simmons PJ, Peault B, Buck DW, Huttner WB
(2000). The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions. J Biol Chem. 275 (8): 5512–20.