KC-4336

293T-STAT5-Luc2-TPOR-Cell-Line

44414

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Background of 293T-STAT5-Luc2-TPOR-Cell-Line

MPL (MPL Proto-Oncogene, Thrombopoietin Receptor) is a Protein Coding gene. Diseases associated with MPL include Myelofibrosis and Thrombocythemia 2. Among its related pathways are Response to elevated platelet cytosolic Ca2+ and Integrin signaling. Gene Ontology (GO) annotations related to this gene include transmembrane signaling receptor activity and thrombopoietin receptor activity.Receptor for thrombopoietin that acts as a primary regulator of megakaryopoiesis and platelet production. May represent a regulatory molecule specific for TPO-R-dependent immune responses.

Specifications

Catalog Number	KC-4336
Cell Line Name	293T-STAT5-Luc2-TPOR-Cell-Line
Host Cell Line	293T-STAT5-Luc2
Description	Stable 293T-STAT5-Luc2 cell line expressing exogenous TPOR 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 +150μg/mL Hygromycin+1μg/mL Puromycin
Selection Marker	Hygromycin and Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 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-STAT5-Luc2-TPOR cell line was generated using lentivirus expressing TPOR sequence.

Characterization

Figure 1: 293T-STAT5-Luc2-TPOR cells were seeded into the 96-well plate, treated with TPO or Romiplostim 6 hours, then readout with Bright-lite™ Luciferase Assay system.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 150μg/mL Hygromycin 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

Germeshausen M, Ballmaier M, Welte K. MPL mutations in 23 patients suffering from congenital amegakaryocytic thrombocytopenia: the type of mutation predicts the course of the disease. Hum Mutat. 2006 Mar;27(3):296. doi: 10.1002/humu.9415. PMID: 16470591.
Bock O, Schlué J, Mengel M, Büsche G, Serinsöz E, Kreipe H. Thrombopoietin receptor (Mpl) expression by megakaryocytes in myeloproliferative disorders. J Pathol. 2004 May;203(1):609-15. doi: 10.1002/path.1558. PMID: 15095485.