KC-1186

CHOK1-TNFRSF1B-Cell-Line

21669

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Background of CHOK1-TNFRSF1B-Cell-Line

TNFRSF1B (Tumor necrosis factor receptor superfamily member 1B), also named as tumor necrosis factor receptor 2 (TNFR2) and CD120b, is a transmembrane protein which binds tumor necrosis factor-alpha (TNF α ). TNFRSF1B is mainly expressed T cells and NK cells, and involved in several autoimmune diseases including Crohn’s disease, systemic lupus erythematosus and familial rheumatoid arthritis.

Specifications

Catalog Number	KC-1186
Cell Line Name	CHOK1-TNFRSF1B-Cell-Line
NCBI/UniProt Accession Number	NM_001066
Host Cell Line	CHOK1
Description	Stable CHOK1 clone expressing exogenous TNFRSF1B 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% RPMI 1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI 1640 + 10% FBS + 10μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:6-1:8 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-TNFRSF1B-cell-line was generated using a lentiviral vector expressing the TNFRSF1B sequence.

Characterization

Figure 1: Characterization of TNFRSF1B overexpression in the CHOK1-TNFRSF1B stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (RPMI 1640 supplemented with 10% FBS and 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 1:6-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI 1640 + 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.Takeuchi, M, M Rothe, and D V Goeddel. 1996. “Anatomy of TRAF2. Distinct Domains for Nuclear Factor-kappaB Activation and Association with Tumor Necrosis Factor Signaling Proteins.” Journal of Biological Chemistry 271 (33): 19935–42.
2.Hijdra, Dani lle, Adriane DM Vorselaars, Jan C Grutters, Anke ME Claessen, and Ger T Rijkers. 2012. “Differential Expression of TNFR1 (CD120a) and TNFR2 (CD120b) on Subpopulations of Human Monocytes.”Journal of Inflammation 9 (1). Journal of Inflammation: 1–1. doi:10.1186/1476-9255-9-38.
3.Bouwmeester, Tewis, Angela Bauch, Heinz Ruffner, Pierre-Olivier Angrand, Giovanna Bergamini, Karen Croughton, Cristina Cruciat, et al. 2004. “A Physical and Functional Map of the Human TNF-Α/NF-κB Signal Transduction Pathway.” Nature Cell Biology 6 (2): 97–105. doi:10.1038/ncb1086.

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.