KC-3793

Ba/F3-NFκB-Luc2 Cell Line

34788

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Background of Ba/F3-NFκB-Luc2 Cell Line

The protein encoded by STAT5 is a member of the STAT family of transcription factors. In response to cytokines and growth factors, STAT family members are phosphorylated by the receptor associated kinases, and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators. It is activated by a wide variety of hematopoietic and nonhematopoietic cytokines and growth factors, all of which use the JAK-STAT signalling pathway as their main mode of signal transduction. It carries out a dual function: signal transduction and activation of transcription. It mediates cellular responses to the cytokine KITLG/SCF and other growth factors. It also mediates cellular responses to ERBB4.

Specifications

Catalog Number	KC-3793
Cell Line Name	Ba/F3-NFκB-Luc2 Cell Line
Clone Number	3B3
Host Cell Line	Ba/F3
Description	Stable Ba/F3 cell line expressing exogenous luciferase gene under the control of STAT5 signaling pathway
Quantity	One vial 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% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS+8ng/mL mIL-3+1mg/mL Hygromycin B
Selection Marker	Hygromycin B
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:10 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 20 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

Ba/F3-STAT5-Luc2 cell line was generated using lentivirus expressing STAT5 sequence.

Characterization

Figure 1. Ba/F3-STAT5-Luc2 cell line was seed into the 96-well plate, and treated with mIL-3 at a maximum concentration 100ng/mL for 6 hours, then readout with Bright-Glo system.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10%FBS, 1mg/mL Hygromycin B, 8ng/mL mIL-3)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 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 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.

References

1.Rani A, Murphy JJ. STAT5 in Cancer and Immunity. J Interferon Cytokine Res. 2016 Apr;36(4):226-37. doi: 10.1089/jir.2015.0054. Epub 2015 Dec 30. PMID: 26716518.
2.Villarino AV, Laurence AD, Davis FP, Nivelo L, Brooks SR, Sun HW, Jiang K, Afzali B, Frasca D, Hennighausen L, Kanno Y, O'Shea JJ. A central role for STAT5 in the transcriptional programing of T helper cell metabolism. Sci Immunol. 2022 Nov 25;7(77):eabl9467. doi: 10.1126/sciimmunol.abl9467. Epub 2022 Nov 25. PMID: 36427325; PMCID: PMC9844264.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.