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	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% 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.

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.