KC-2620

Ba/F3-KRAS-Q61H-Cell-Line

44822

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Background of Ba/F3-KRAS-Q61H-Cell-Line

K-ras was the first identified oncogene in cellular genome, KRAS protein can bind GDP/GTP and possess intrinsic GTPase activity, plays an important role in the regulation of cell proliferation, the overactivation due to mutation can lead to continuously cell proliferation, and eventually develop into cancer. Ba/F3 cell, a murine interleukin-3 dependent pro-B cell line, is a popular system for exploring both kinases and their inhibitors, because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects.

Specifications

Catalog Number	KC-2620
Cell Line Name	Ba/F3-KRAS-Q61H-Cell-Line
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous KRAS bearing Q61H amino acid 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
In Vivo Validation	NA

Cell Line Generation

Ba/F3 KRAS-Q61H cell Line was generated using retrovirus vector expressing human KRAS-Q61H sequence.

Characterization

Figure1: Characterization of KRAS and its mutants overexpressing in Ba/F3 stable clones using PCR sequencing

Figure2: 1.Harvest and seed the Ba/F3 cells expressing KRAS mutant in 96-well plate (3000 cells/90ul medium). 2.Next day, add 10ul 10X serially diluted compound solution each well and incubate the plates for another 72 hours. 3.Add 100ul Cell Titer-Glo each well, mixed and readout using Envision. 4.Plot the dose-responsive curve and fit the IC50 (the centration of 50% inhibition of DMSO vehicle treated clones) using GraphPad Prism software (Version 5).

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 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

Suchida N, Ryder T, Ohtsubo E (1982). Nucleotide sequence of the oncogene encoding p21 transforming protein of Kirsten murine sarcoma virus. Science. 217 (4563): 937–939.
Ostrem, Jonathan M, Ulf Peters, Martin L Sos, James A Wells, and Kevan M Shokat. 2013. “K-Ras(G12C) Inhibitors Allosterically Control GTP Affinity and Effector Interactions.” Nature 503 (7477). Nature Publishing Group: 548–51. doi:10.1038/nature12796.
Janes, Matthew R, Jingchuan Zhang, Lian-Sheng Li, Rasmus Hansen, Ulf Peters, Xin Guo, Yuching Chen, et al. 2018. “Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor.” Cell 172 (3). Elsevier Inc.: 578–581.e17.