KC-5407

A204-SMAD-Luc2

61013

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Background of A204-SMAD-Luc2

Transforming growth factor-β1 (TGF-β1) is considered as a crucial mediator in tissue fibrosis and causes tissue scarring largely by activating its downstream small mother against decapentaplegic (SMAD) signaling. Different TGF-β signalings play different roles in fibrogenesis. TGF-β1 directly activates Smad signaling which triggers pro-fibrotic gene overexpression. Smad transcription factors lie at the core of one of the most versatile cytokine signaling pathways in metazoan biology-the transforming growth factor-beta (TGFbeta) pathway. Recent progress has shed light into the processes of Smad activation and deactivation, nucleocytoplasmic dynamics, and assembly of transcriptional complexes. A rich repertoire of regulatory devices exerts control over each step of the Smad pathway.

Specifications

Catalog Number	KC-5407
Cell Line Name	A204-SMAD-Luc2
Clone Number	1B1
Host Cell Line	A204
Description	Stable A204 cell line expressing exogenous luciferase gene under the control of SMAD 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% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS+150µg/mL Hygromycin B
Selection Marker	Hygromycin B
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

Cell Line Generation

A204-SMAD-Luc2 Cell Line was generated using a lentiviral vector expressing SMAD sequence.

Characterization

Figure 1. A204-SMAD-Luc2 cell line was seed into the 96-well plate, and treated with Activin A at a maximum concentration 1000 ng/mL diluted 3.16-fold for 16 hours, then readout with Bright-Glo system.

Figure 2. A204-SMAD-Luc2 cell line was seed into the 96-well plate, and treated with GDF-8 at a maximum concentration 1000 ng/mL diluted 3.16-fold for 16 hours, then readout with Bright-Glo system.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS, and 150µg/mL Hygromycin B)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 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 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. Hu HH, Chen DQ, Wang YN, Feng YL, Cao G, Vaziri ND, Zhao YY. New insights into TGF-β/Smad signaling in tissue fibrosis. Chem Biol Interact. 2018 Aug 25;292:76-83. doi: 10.1016/j.cbi.2018.07.008. Epub 2018 Jul 11. PMID: 30017632.
2. Massagué J, Seoane J, Wotton D. Smad transcription factors. Genes Dev. 2005 Dec 1;19(23):2783-810. doi: 10.1101/gad.1350705. PMID: 16322555.