KC-4284

293T-SMAD-Luc2-ACVR2A-KO-ACVR2B-Cell-Line

44086

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Background of 293T-SMAD-Luc2-ACVR2A-KO-ACVR2B-Cell-Line

Activins are members of the TGF-β family of ligands that have multiple biological functions in embryonic stem cells as well as in differentiated tissue. Serum levels of activin A were found to be elevated in pathological conditions such as cachexia, osteoporosis and cancer. Smads are intracellular proteins that act as central effectors for transforming growth factor-beta (TGF-beta) and related proteins from the activated receptor into the nucleus, where they regulate ligand-induced gene expression.Smad4 is a central mediator for TGF-β pathway.The phosphorylated form of this protein forms a complex with SMAD4, which is important for its function in the transcription regulation.

Specifications

Catalog Number	KC-4284
Cell Line Name	293T-SMAD-Luc2-ACVR2A-KO-ACVR2B-Cell-Line
NCBI/UniProt Accession Number	NM_001106
Clone Number	1A2
Host Cell Line	293T-SMAD-Luc2-ACVR2A-ACVR2B-KO
Description	Stable 293T cell line expressing exogenous luciferase gene under the control of ACVR2B signaling pathway with human ACVR2A and ACVR2B gene knockout.
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+1μg/mL puromycin
Selection Marker	Puromycin \| Hygromycin B
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 every 2-3 days
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T-SMAD-Luc2-ACVR2A-KO-ACVR2B cell line was generated using lentivirus expressing ACVR2B sequence.

Characterization

Figure 1. 293T-SMAD-Luc2-ACVR2A-KO-ACVR2B cell line was seeded into the 96-well plate, and treated with TGFβ1 and Activin A at a maximum concentration of 100ng/mL for 16 hours, then readout with Bright-Glo system.

Cell Resuscitation

Pre-warm complete culture medium (basal medium and 10% FBS) in a 37°C water bath.
Rapidly thaw the cryovial in a 37°C water bath for 1-2 minutes with gentle agitation.
Transfer the vial to a biosafety cabinet, and disinfect the exterior with 70% ethanol.
Aseptically transfer the cell suspension dropwise into a sterile centrifuge tube containing 9.0 mL of pre-warmed complete medium.
Centrifuge at approximately 125 × g for 5–7 minutes at room temperature, carefully aspirate the supernatant without disturbing the cell pellet.
Gently resuspend the pellet in an appropriate volume of complete medium and transfer the suspension into a T25 flask.
Incubate the flask in a 37°C in a humidified 5% CO₂ incubator.
Assess cell viability and morphology after 24 hours. If cells appear healthy, replace the medium with fresh medium supplemented with the appropriate selective antibiotic.
Subculture the cells at a ratio of 1:4-1:8 every 2-3 days upon reaching 80%–90% confluency.

Cell Freezing

Prepare the freezing medium (70% basal medium, 20% FBS and 10% DMSO) freshly before use.
Pre-chill the freezing medium on ice and label the cryovials accordingly.
Transfer the cell suspension to a sterile conical tube and perform a cell count to determine total viability and density.
Centrifuge the cells at 250×g for 5 minutes at room temperature; carefully aspirate the supernatant.
Gently resuspend the cell pellet in chilled freezing medium, ensuring a minimum cell density of 3×10⁶ cells/mL.
Aliquot 1 mL of the cell suspension into each pre-labeled cryovial.
Place the cryovials into a CoolCell® container and store at -80°C overnight for controlled-rate cooling.
Transfer the cryovials to the liquid nitrogen for long-term storage the following day.

References

Olsen OE, Wader KF, Hella H, Mylin AK, Turesson I, Nesthus I, Waage A, Sundan A, Holien T. Activin A inhibits BMP-signaling by binding ACVR2A and ACVR2B. Cell Commun Signal. 2015 Jun 6;13:27. doi: 10.1186/s12964-015-0104-z. PMID: 26047946; PMCID: PMC4467681.
Bai L, Chang HM, Cheng JC, Chu G, Leung PCK, Yang G. ALK2/ALK3-BMPR2/ACVR2A Mediate BMP2-Induced Downregulation of Pentraxin 3 Expression in Human Granulosa-Lutein Cells. Endocrinology. 2017 Oct 1;158(10):3501-3511. doi: 10.1210/en.2017-00436. PMID: 28977600.

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.