KC-0316

293T-cyno-CD123-Cell-Line

20217

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Background of 293T-cyno-CD123-Cell-Line

CD123, also known as interleukin-3 receptor, belongs to the type I cytokine receptor family, and form a heterodimer with common beta (CD131) subunit, which transmit the signal after binding with interleukin-3. CD123 is mainly found on pluripotent progenitor cells, and also many subtype of acute myeloid leukemia (AML), and is a potential target of treatment for AML.

Specifications

Catalog Number	KC-0316
Cell Line Name	293T-cyno-CD123-Cell-Line
Host Cell Line	Human HEK293T cell line
Description	HEK293T cell line stable expressing exogenous cynomolgus CD123 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 cyno CD123 cell line was generated using lentiviral vector expressing cynomolgus CD123 sequence.

Characterization

Figure: Characterization of CD123 overexpressing in 293T stable clones using FACS.

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

Testa, U., Pelosi, E. & Frankel, A. CD 123 is a membrane biomarker and a therapeutic target in hematologic malignancies. Biomark Res 2, 4 (2014).
Ehninger, A. et al. Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia. 4, e218ÿ10 (2014).
Nievergall, E. et al. Monoclonal antibody targeting of IL-3 receptor ϫ with CSL362 effectively depletes CML progenitor and stem cells. Blood 123, 1218ÿ1228 (2014).