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	One vial 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.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.