KC-5096

Ba/F3-AKT2-E17K Cell Line

57221

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Background of Ba/F3-AKT2-E17K Cell Line

AKT2 is a putative oncogene encoding a protein belonging to a subfamily of serine/threonine kinases containing SH2-like (Src homology 2-like) domains, which is involved in signaling pathways. The gene serves as an oncogene in the tumorigenesis of cancer cells For example, its overexpression contributes to the malignant phenotype of a subset of human ductal pancreatic cancers. The encoded protein is a general protein kinase capable of phophorylating several known proteins, and has also been implicated in insulin signaling.

Specifications

Catalog Number	KC-5096
Cell Line Name	Ba/F3-AKT2-E17K Cell Line
Clone Number	1-8#
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous AKT2 gene bearing E17K mutation.
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% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+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

Cell Line Generation

Ba/F3 AKT2 E17K cell line was generated using retrovirus vector expressing human AKT2 E17K sequence.

Characterization

Figure 1: Characterization of AKT2 mutation in the Ba/F3 stable clone using PCR sequencing.

Figure 2: Ba/F3 cells expressing AKT2 mutant were seeded into 96-well plates, treated with compounds for 72 hours, and then read out with Cell-Titer Glo system.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 10% FBS)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% RPMI-1640 + 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. Ghosh S, Sharma R, Bammidi S, Koontz V, Nemani M, Yazdankhah M, Kedziora KM, Stolz DB, Wallace CT, Yu-Wei C, Franks J, Bose D, Shang P, Ambrosino HM, Dutton JR, Geng Z, Montford J, Ryu J, Rajasundaram D, Hose S, Sahel JA, Puertollano R, Finkel T, Zigler JS Jr, Sergeev Y, Watkins SC, Goetzman ES, Ferrington DA, Flores-Bellver M, Kaarniranta K, Sodhi A, Bharti K, Handa JT, Sinha D. The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in non-neovascular AMD. Nat Commun. 2024 Jul 21;15(1):6150.