KC-4449

CHOK1-cyno-CD70 Cell Line

49273

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Background of CHOK1-cyno-CD70 Cell Line

CD70, The protein encoded by this gene is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. This cytokine is a ligand for TNFRSF27/CD27. It is a surface antigen on activated, but not on resting, T and B lymphocytes. It induces proliferation of costimulated T cells, enhances the generation of cytolytic T cells, and contributes to T cell activation. This cytokine is also reported to play a role in regulating B-cell activation, cytotoxic function of natural killer cells, and immunoglobulin sythesis.

Specifications

Catalog Number	KC-4449
Cell Line Name	CHOK1-cyno-CD70 Cell Line
Host Cell Line	CHOK1
Description	CHOK1 cell line stably expressing exogenous cyno CD70 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 + 10 μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Fibroblastoid cells growing as monolayer
Subculture	Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-2 × 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

CHOK1-cyno-CD70 cell line was generated using a lentiviral vector expressing the cyno CD70 sequence.

Characterization

Figure1: Characterization of cyno CD70 overexpression in CHOK1 cyno CD70 stable clones using quantitative real-time PCR.

Figure2: Characterization of CHOK1-cyno-CD70 cell line stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10 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 9.0 mL 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 1-2 × 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.Yu SE, Park SH, Jang YK. Epigenetic silencing of TNFSF7 (CD70) by DNA methylation during progression to breast cancer. Mol Cells. 2010 Feb 28;29(2):217-21. doi: 10.1007/s10059-010-0052-9. Epub 2010 Jan 28. PMID: 20119871.
2.Pratt D, Pittaluga S, Palisoc M, Fetsch P, Xi L, Raffeld M, Gilbert MR, Quezado M. Expression of CD70 (CD27L) Is Associated With Epithelioid and Sarcomatous Features in IDH-Wild-Type Glioblastoma. J Neuropathol Exp Neurol. 2017 Aug 1;76(8):697-708. doi: 10.1093/jnen/nlx051. PMID: 28789475; PMCID: PMC6251514.
3.Aftimos P, Rolfo C, Rottey S, Offner F, Bron D, Maerevoet M, Soria JC, Moshir M, Dreier T, Van Rompaey L, Michot JM, Silence K, Hultberg A, Gandini D, de Haard H, Ribrag V, Peeters M, Thibault A, Leupin N, Awada A. Phase I Dose-Escalation Study of the Anti-CD70 Antibody ARGX-110 in Advanced Malignancies. Clin Cancer Res. 2017 Nov 1;23(21):6411-6420. doi: 10.1158/1078-0432.CCR-17-0613. Epub 2017 Aug 1. PMID: 28765328.