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	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% 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.