KC-2555

293T-OS8-VISTA-Cell-Line

24294

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Background of 293T-OS8-VISTA-Cell-Line

T cell-activated V structural domain Ig suppressor (VISTA) is a novel immunomodulatory receptor that is widely expressed on myeloid and lymphoid cells, and is often considered an indicator of poor prognosis in a variety of cancers. Importantly, antibody targeting of VISTA acts on both intrinsic and adaptive immunity.The widespread expression of VISTA gives it a higher non-redundant activity compared to other immune checkpoints, making VISTA a very promising target for tumor immunotherapy.

Specifications

Catalog Number	KC-2555
Cell Line Name	293T-OS8-VISTA-Cell-Line
Host Cell Line	293T-OS8
Description	Stable 293T OS8 clone expressing exogenous VISTA 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+150µg/mL Hygromycin B
Selection Marker	Puromycin and Hygromycin B
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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-OS8-VISTA cell line was generated using a lentiviral vector expressing the VISTA sequence.

Characterization

Figure 1: Characterization of VISTA overexpression in the 293T-OS8-VISTA stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 150µg/mL Hygromycin B and 1μg/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 1:4-1:8 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

Yuan L, Tatineni J, Mahoney KM, Freeman GJ. VISTA: A Mediator of Quiescence and a Promising Target in Cancer Immunotherapy. Trends Immunol. 2021 Mar;42(3):209-227. doi: 10.1016/j.it.2020.12.008. Epub 2021 Jan 23. PMID: 33495077; PMCID: PMC8088836.
Martin AS, Molloy M, Ugolkov A, von Roemeling RW, Noelle RJ, Lewis LD, Johnson M, Radvanyi L, Martell RE. VISTA expression and patient selection for immune-based anticancer therapy. Front Immunol. 2023 Feb 20;14:1086102. doi: 10.3389/fimmu.2023.1086102. PMID: 36891296; PMCID: PMC9986543.
ElTanbouly MA, Zhao Y, Schaafsma E, Burns CM, Mabaera R, Cheng C, Noelle RJ. VISTA: A Target to Manage the Innate Cytokine Storm. Front Immunol. 2021 Feb 11;11:595950. doi: 10.3389/fimmu.2020.595950. PMID: 33643285; PMCID: PMC7905033.