KC-3894

CHOK1-mouse-VISTA Cell Line

34984

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Background of CHOK1-mouse-VISTA Cell Line

VISTA also belongs to the B7 family of molecules and shows significant homology to PD-L1 and PD-L2. Unlike other immune checkpoint molecules, VISTA is constitutively expressed on naïve T cells, which are lost during the immune response, and on the surface of antigen-presenting cells in addition to T cells, and inhibits T cell activation through both intra- and extra-T cellular mechanisms. In short, VISTA is a unique negative checkpoint regulator on initial T-cells and is essential for maintaining their resting and peripheral immune tolerance steady state.

Specifications

Catalog Number	KC-3894
Cell Line Name	CHOK1-mouse-VISTA Cell Line
NCBI/UniProt Accession Number	NM_028732.4
Clone Number	4#
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous mouse 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 10μg/mL Puromycin
Selection Marker	Puromycin
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

CHOK1-mouse-VISTA cell line was generated using a lentiviral vector expressing the mouse VISTA sequence.

Characterization

Figure 1: Characterization of mouse VISTA overexpression in the CHOK1-mouse-VISTA stable clone using FACS.

Figure 2: Characterization of mouse VISTA in the CHOK1 stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 10% FBS + 10μg/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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 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. Mulati, Kumuluzi, Junzo Hamanishi, Noriomi Matsumura, Kenji Chamoto, Nathan Mise, Kaoru Abiko, Tsukasa Baba et al. 2019. VISTA expressed in tumour cells regulates T cell function. British journal of cancer 120(1):115-127.
2. Gabr, Moustafa T., and Sanjiv S. Gambhir. 2020. Discovery and Optimization of Small-Molecule Ligands for V-Domain Ig Suppressor of T-Cell Activation (VISTA). Journal of the American Chemical Society 142(38):16194-16198.
3. ElTanbouly, Mohamed A., Yanding Zhao, Elizabeth Nowak, Jiannan Li, Evelien Schaafsma, Isabelle Le Mercier, Sabrina Ceeraz et al. 2020. VISTA is a checkpoint regulator for naive T cell quiescence and peripheral tolerance. Science 367(6475).

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.