KC-1006

293T-Human-IDO1-Cell-Line

43797

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Background of 293T-Human-IDO1-Cell-Line

"Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the first and rate-limiting step in tryptophan catabolism to N-formyl-kynurenine. This enzyme is thought to play a role in a variety of pathophysiological processes such as antimicrobial and antitumor defense, neuropathology, immunoregulation, and antioxidant activity. Through its expression in dendritic cells, monocytes, and macrophages this enzyme modulates T-cell behavior by its peri-cellular catabolization of the essential amino acid tryptophan. "

Specifications

Catalog Number	KC-1006
Cell Line Name	293T-Human-IDO1-Cell-Line
Host Cell Line	293T
Description	HEK293T cell line stably expressing exogenous human IDO1 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 + 1 μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Fibroblastoid cells growing as monolayer
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 human IDO1 cell line was generated using lentiviral vector expressing human IDO1 sequence.

Characterization

Figure 1: Characterization of IDO1 overexpressing in 293T stable clones using cellular enzymatic assay.

Figure 1: hIDO1 inhibitors screening with cellular assay.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 1 μg/mL puromycin)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 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 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

Liu, Xiangdong et al. Selective inhibition of IDO1 effectively regulates mediators of antitumor immunity. Blood 115.17 (2010): 3520-3530. Web. 27 April. 2018.
Soliman, Hatem, Melanie Mediavilla-Varela, and Scott Antonia. Indoleamine 2,3-Dioxygenase: Is It an Immune Suppressor? Cancer journal (Sudbury, Mass.) 16.4 (2010): 10.1097/PPO.0b013e3181eb3343. PMC. Web. 28 Apr. 2018.