KC-5132

CHOK1-mouse-BAFFR cell line

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Background of CHOK1-mouse-BAFFR cell line

The B lymphocyte stimulator receptor (BAFFR, also known as TNFRSF13C, BROMIX, CD268, CVID4, prolixin) is encoded by the TNFRSF13C gene and is one of the main membrane protein receptors for B cell proliferation and differentiation. It belongs to the TNF receptor (TNFR) superfamily, but BAFFR differs from other TNFRs in that its CRDs (cysteine-rich domains) contain only four cysteine residues, which are used for ligand binding and self-assembly.

Specifications

Catalog Number	KC-5132
Cell Line Name	CHOK1-mouse-BAFFR cell line
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous mouse BAFFR 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 1:4-1:8 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

Cell Line Generation

CHOK1 mouse-BAFFR cell line was generated using lentiviral vector expressing mouse-BAFFR sequence

Characterization

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

Figure 2: Characterization of mouse-BAFFR overexpression in the CHOK1 mouse-BAFFR stable clone using PCR sequence

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 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 1:4-1:8 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. Ntellas P, Dardiotis E, Sevdali E, Siokas V, Aloizou AM, Tsinti G, Germenis AE, Hadjigeorgiou GM, Eibel H, Speletas M. TNFRSF13C/BAFFR P21R and H159Y polymorphisms in multiple sclerosis. Mult Scler Relat Disord. 2020 Jan;37:101422. doi: 10.1016/j.msard.2019.101422. Epub 2019 Sep 30. PMID: 32172995.
2.Prieto-Peña D, Genre F, Remuzgo-Martínez S, Pulito-Cueto V, Atienza-Mateo B, Llorca J, Sevilla-Pérez B, Ortego-Centeno N, Lera-Gómez L, Leonardo MT, Peñalba A, Narváez J, Martín-Penagos L, Rodrigo E, Miranda-Filloy JA, Caminal-Montero L, Collado P, Pérez JS, de Argila D, Rubio E, Luque ML, Blanco-Madrigal JM, Galíndez-Agirregoikoa E, Gualillo O, Martín J, Castañeda S, Blanco R, González- Gay MA, López-Mejías R. BAFF, APRIL and BAFFR on the pathogenesis of Immunoglobulin-A vasculitis. Sci Rep. 2021 Jun 1;11(1):11510. doi: 10.1038/s41598-021-91055-z. Erratum in: Sci Rep. 2021 Aug 16;11(1):16946. doi: 10.1038/s41598-021-96114-z. PMID: 34075170; PMCID: PMC8169776.
3.Müller-Winkler J, Mitter R, Rappe JCF, Vanes L, Schweighoffer E, Mohammadi H, Wack A, Tybulewicz VLJ. Critical requirement for BCR, BAFF, and BAFFR in memory B cell survival. J Exp Med. 2021 Feb 1;218(2):e20191393. doi: 10.1084/jem.20191393. PMID: 33119032; PMCID: PMC7604764.