KC-3249

293T-CAIX Cell Line

34250

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Background of 293T-CAIX Cell Line

Carbonic anhydrase 9 (CAIX) is a transmembrane metalloenzyme that regulates cellular adhesion, proliferation, and intra/extracellular pH. It is over-expressed in renal cell carcinoma. CAIX to be an essential part of the tumour microenvironment and a possible master regulator of tumour progression. This makes CAIX a highly effective and feasible therapeutic target for selective cancer treatment.

Specifications

Catalog Number	KC-3249
Cell Line Name	293T-CAIX Cell Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous CAIX 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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
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
In Vivo Validation	NA

Cell Line Generation

293T CAIX cell line was generated using a lentiviral vector expressing the CAIX sequence.

Characterization

Figure 1: Characterization of CAIX overexpression in 293T stable clones using FACS.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/mL puromycin)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. Pérez-Sayáns M, Suárez-Peñaranda JM, Pilar GD, Supuran CT, Pastorekova S, Barros-Angueira F, Gándara-Rey JM, García-García A. Expression of CA-IX is associated with advanced stage tumors and poor survival in oral squamous cell carcinoma patients. J Oral Pathol Med. 2012 Oct;41(9):667-74. doi: 10.1111/j.1600-0714.2012.01147.x. Epub 2012 Apr 4. PMID: 22486898. 2. Tu C, Foster L, Alvarado A, McKenna R, Silverman DN, Frost SC. Role of zinc in catalytic activity of carbonic anhydrase IX. Arch Biochem Biophys. 2012 May;521(1-2):90-4. doi: 10.1016/j.abb.2012.03.017. Epub 2012 Mar 23. PMID: 22465027; PMCID: PMC3345035.