KC-6442

E0771-BAFFR-low Cell Line

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Background of E0771-BAFFR-low Cell Line

B cell-activating factor (BAFF) enhances B-cell survival in vitro and is a regulator of the peripheral B-cell population. Overexpression of Baff in mice results in mature B-cell hyperplasia and symptoms of systemic lupus erythematosus (SLE). Also, some SLE patients have increased levels of BAFF in serum. Therefore, it has been proposed that abnormally high levels of BAFF may contribute to the pathogenesis of autoimmune diseases by enhancing the survival of autoreactive B cells. The protein encoded by this gene is a receptor for BAFF and is a type III transmembrane protein containing a single extracellular cysteine-rich domain. It is thought that this receptor is the principal receptor required for BAFF-mediated mature B-cell survival.

Specifications

Catalog Number	KC-6442
Cell Line Name	E0771-BAFFR-low Cell Line
NCBI/UniProt Accession Number	NM_052945.4
Clone Number	9#
Host Cell Line	E0771
Description	E0771 cell line stably expressing exogenous 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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 2 μg/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

E0771-BAFFR-low cell line was generated using a lentiviral vector expressing the BAFFR sequence.

Characterization

Figure1: Characterization of BAFFR overexpressing in E0771 stable clones using FACS.Expression levels were quantified by qFACS at approximately 4408 molecules per cell.

Figure2: Characterization of E0771-BAFFR-low cell line stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 2 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 9.0mL 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% DMEM + 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

Mihalcik SA, Huddleston PM 3rd, Wu X, Jelinek DF. The structure of the TNFRSF13C promoter enables differential expression of BAFF-R during B cell ontogeny and terminal differentiation. J Immunol. 2010 Jul 15;185(2):1045-54. doi: 10.4049/jimmunol.1001120. Epub 2010 Jun 16. Erratum in: J Immunol. 2010 Nov 15;185(10):6384. PMID: 20554963; PMCID: PMC3780588.
Min YN, Wang CY, Li XX, Hou Y, Qiu JH, Ma J, Shao LL, Zhang X, Wang YW, Peng J, Hou M, Shi Y. Participation of B-cell-activating factor receptors in the pathogenesis of immune thrombocytopenia. J Thromb Haemost. 2016 Mar;14(3):559-71. doi: 10.1111/jth.13246. Epub 2016 Feb 17. PMID: 26749059.
Lougaris V, Baronio M, Moratto D, Cardinale F, Plebani A. Monoallelic BAFFR P21R/H159Y Mutations and Familiar Primary Antibody Deficiencies. J Clin Immunol. 2016 Jan;36(1):1-3. doi: 10.1007/s10875-015-0217-6. Epub 2015 Nov 27. PMID: 26613719.