KC-5693

293T-MECA447-P Cell Line

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Background of 293T-MECA447-P Cell Line

CD19 (CD19 Molecule) is a Protein Coding gene. This gene encodes a member of the immunoglobulin gene superfamily. CD19 is a B-cell transmembrane molecule that is critical for B-cell activation. Expression of this cell surface protein is restricted to B cell lymphocytes. This protein is a reliable marker for pre-B cells but its expression diminishes during terminal B cell differentiation in antibody secreting plasma cells. Activation of B lymphocytes involves binding of antigen to the specific receptor and signalling through several membrane coreceptors, of which CD19 has been found to play a pivotal role as a response regulator. MECA447 is an autoimmune-associated antigen, MECA447 may be related to autoantigens associated with vascular endothelial cells and is often used in rheumatology and immunology research. Diseases associated with CD19 include Immunodeficiency, Common Variable, 3 and Common Variable Immunodeficiency Phenotype Due To Cd19/Cd81 Deficiency.

Specifications

Catalog Number	KC-5693
Cell Line Name	293T-MECA447-P Cell Line
Clone Number	3#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous human MECA447-P 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 + 150μg/mL Hygromycin B
Selection Marker	Hygromycin B
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

293T-human-MECA447-P cell line was generated using a lentiviral vector expressing the human MECA447-P sequence.

Characterization

Figure 1: Characterization of mouse NRP1 overexpression in the 293T human MECA447-P stable clone using FACS(Kyinno, Cat#KP-1123).

Figure 2: Characterization of human MECA447-P in the 293T stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 150μg/mL Hygromycin B)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% DMEM + 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. Fujimoto M, Poe JC, Jansen PJ, Sato S, Tedder TF. CD19 amplifies B lymphocyte signal transduction by regulating Src-family protein tyrosine kinase activation. J Immunol. 1999 Jun 15;162(12):7088-94. PMID: 10358152.
2. Gärdby E, Chen XJ, Lycke NY. Impaired CD40-signalling in CD19-deficient mice selectively affects Th2-dependent isotype switching. Scand J Immunol. 2001 Jan;53(1):13-23. doi: 10.1046/j.1365-3083.2001.00824.x. PMID: 11169202.
3. Kong D, Kwon D, Moon B, Kim DH, Kim MJ, Choi J, Kang KS. CD19 CAR-expressing iPSC-derived NK cells effectively enhance migration and cytotoxicity into glioblastoma by targeting to the pericytes in tumor microenvironment. Biomed Pharmacother. 2024 May;174:116436. doi: 10.1016/j.biopha.2024.116436. Epub 2024 Mar 19. PMID: 38508081.