KC-4033

Ba/F3-NFκB-GFP Cell Line

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Background of Ba/F3-NFκB-GFP Cell Line

NF-κB pathway consists of canonical and non-canonical pathways. The canonical NF-κB is activated by various stimuli, transducing a quick but transient transcriptional activity, to regulate the expression of various proinflammatory genes and also serve as the critical mediator for inflammatory response. Meanwhile, the activation of the non-canonical NF-κB pathway occurs through a handful of TNF receptor superfamily members.

Specifications

Catalog Number	KC-4033
Cell Line Name	Ba/F3-NFκB-GFP Cell Line
Clone Number	10A4
Host Cell Line	Ba/F3
Description	Stable Ba/F3 cell line expressing exogenous green fluorescent protein gene under the control of NFκB 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-NFκB-GFP cell line was generated using lentivirus expressing NFκB sequence.

Characterization

Figure 1. Ba/F3-NFκB-GFP cell line was seed into the 96-well plate, and treated with mTNF-α at a maximum concentration 1μg/mL for 6 hours, then readout with FACS.

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.Yu H, Lin L, Zhang Z, Zhang H, Hu H. Targeting NF-κB pathway for the therapy of diseases: mechanism and clinical study. Signal Transduct Target Ther. 2020 Sep 21;5(1):209. doi: 10.1038/s41392-020-00312-6. PMID: 32958760; PMCID: PMC7506548.
2.Lawrence T. The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb Perspect Biol. 2009 Dec;1(6):a001651. doi: 10.1101/cshperspect.a001651. Epub 2009 Oct 7. PMID: 20457564; PMCID: PMC2882124.

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.