KC-2210

CHO-K1-human-PVRL2-Middle-Cell-Line

47050

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Background of CHO-K1-human-PVRL2-Middle-Cell-Line

NECTIN2 (Nectin Cell Adhesion Molecule 2) is a Protein Coding gene. Diseases associated with NECTIN2 include Herpes Simplex and Multiple Sclerosis. Among its related pathways are Cell junction organization and Sertoli-Sertoli Cell Junction Dynamics. An important paralog of this gene is PVR.

Specifications

Catalog Number	KC-2210
Cell Line Name	CHO-K1-human-PVRL2-Middle-Cell-Line
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous human PVRL2-Middle 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 10μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 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

Cell Line Generation

CHO-K1-human-PVRL2-Middle Cell Line was generated using a lentiviral vector expressing the human PVRL2-Middle sequence.

Characterization

Figure 1: Characterization of human-PVRL2-Middle overexpression in the CHO-K1 human-PVRL2-Middle stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10μg/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% RPMI1640 + 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

1: Yang J, Wang L, Byrnes JR, Kirkemo LL, Driks H, Belair CD, Aguilar OA, Lanier
LL, Wells JA, Fong L, Blelloch R. PVRL2 Suppresses Antitumor Immunity through
PVRIG- and TIGIT-independent Pathways. Cancer Immunol Res. 2024 May
2;12(5):575-591. doi: 10.1158/2326-6066.CIR-23-0722. PMID: 38588410; PMCID:
PMC11063765.
2: Byrnes JR, Weeks AM, Shifrut E, Carnevale J, Kirkemo L, Ashworth A, Marson A,
Wells JA. Hypoxia Is a Dominant Remodeler of the Effector T Cell Surface
Proteome Relative to Activation and Regulatory T Cell Suppression. Mol Cell
Proteomics. 2022 Apr;21(4):100217. doi: 10.1016/j.mcpro.2022.100217. Epub 2022
Feb 23. PMID: 35217172; PMCID: PMC9006863.
3: Elledge SK, Zhou XX, Byrnes JR, Martinko AJ, Lui I, Pance K, Lim SA, Glasgow
JE, Glasgow AA, Turcios K, Iyer NS, Torres L, Peluso MJ, Henrich TJ, Wang TT,
Tato CM, Leung KK, Greenhouse B, Wells JA. Engineering luminescent biosensors
for point-of-care SARS-CoV-2 antibody detection. Nat Biotechnol. 2021
Aug;39(8):928-935. doi: 10.1038/s41587-021-00878-8. Epub 2021 Mar 25. PMID:
33767397; PMCID: PMC8355051.