KC-5564

Ba/F3-AKT1-Flag Cell Line

59778

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Background of Ba/F3-AKT1-Flag Cell Line

AKT1 (AKT Serine/Threonine Kinase 1), also known as PKB (Protein Kinase B), RAC-alpha, or PRKBA, is a key serine/threonine kinase and a core component of the PI3K-AKT-mTOR signaling pathway. It regulates critical cellular processes including proliferation, survival, metabolism, and angiogenesis. AKT1 is ubiquitously expressed, with high levels in adipose tissue, lung, and aorta. As a well-established oncogene, AKT1 is implicated in various cancers, including breast, colorectal, ovarian, and pancreatic cancers. The most common activating mutation, E17K, is found in multiple solid tumors. Drug development has evolved from pan-AKT inhibitors (e.g., capivasertib) to mutation-selective inhibitors like ALTA2618, which target AKT1 E17K to improve efficacy and reduce toxicity.

Specifications

Catalog Number	KC-5564
Cell Line Name	Ba/F3-AKT1-Flag Cell Line
NCBI/UniProt Accession Number	NM_005163
Clone Number	2#
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous human AKT1 and Flag gene.
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% RPMI 1640+20% FBS+10% DMSO
Propagation Medium	RPMI 1640+10%FBS
Selection Marker	Puromycin
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-AKT1-Flag cell Line was generated using retrovirus vector expressing human AKT1 and Flag sequence.

Characterization

Figure 1: Ba/F3-AKT1-Flag cell were seeded into 96-well plates, treated with compounds for 72 hours, and then read out with Cell-Titer Glo system.

Figure 2: Characterization of AKT1 in the Ba/F3-AKT1-Flag stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (RPMI 1640 + 10% FBS) in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI 1640 + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage.

References

Tian X, Wang R, Gu T, Ma F, Laster KV, Li X, Liu K, Lee MH, Dong Z. Costunolide is a dual inhibitor of MEK1 and AKT1/2 that overcomes osimertinib resistance in lung cancer. Mol Cancer. 2022 Oct 6;21(1):193. doi: 10.1186/s12943-022-01662-1. PMID: 36203195; PMCID: PMC9535870.