KC-5627

K562-CD19-GFP-Luc2 Cell Line

60622

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Background of K562-CD19-GFP-Luc2 Cell Line

This gene encodes a member of the immunoglobulin gene superfamily. 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. The protein has two N-terminal extracellular Ig-like domains separated by a non-Ig-like domain, a hydrophobic transmembrane domain, and a large C-terminal cytoplasmic domain. This protein forms a complex with several membrane proteins including complement receptor type 2 (CD21) and tetraspanin (CD81) and this complex reduces the threshold for antigen-initiated B cell activation. Activation of this B-cell antigen receptor complex activates the phosphatidylinositol 3-kinase signalling pathway and the subsequent release of intracellular stores of calcium ions. This protein is a target of chimeric antigen receptor (CAR) T-cells used in the treatment of lymphoblastic leukemia. Mutations in this gene are associated with the disease common variable immunodeficiency 3 (CVID3) which results in a failure of B-cell differentiation and impaired secretion of immunoglobulins. CVID3 is characterized by hypogammaglobulinemia, an inability to mount an antibody response to antigen, and recurrent bacterial infections. Alternative splicing results in multiple transcript variants encoding distinct isoforms.
Luciferase is an oxidative enzyme that can produce bioluminescence with the addition of luciferin, but don’t need an external light source, which is different from fluorescent proteins. The bioluminescence can be detected directly by light sensitive device, such as luminometer or modified microscope.Luciferase is widely used in many fields of biological research, such as transcriptional activity, kinase or other enzyme activity, cellular ATP level, whole animal imaging.
Green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range.

Specifications

Catalog Number	KC-5627
Cell Line Name	K562-CD19-GFP-Luc2 Cell Line
NCBI/UniProt Accession Number	NM_001770.5
Clone Number	4#
Host Cell Line	K562-GFP-Luc2
Description	Stable K562-GFP-Luc2 cell line expressing exogenous CD19 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 + 1μg/mL Puromycin + 300μg/mL Hygromycin B
Selection Marker	Puromycin \| Hygromycin B
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:4 every 2 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

K562-CD19-GFP-Luc2 cell line was generated using a lentiviral vector expressing the CD19 sequence.

Characterization

Figure 1: Characterization of CD19 overexpression in the K562-CD19-GFP-Luc2 stable clone using FACS.

Figure 2: Characterization of GFP overexpression in the K562-CD19-GFP-Luc2 stable clone using FACS.

Figure 3: Characterization of luciferase expression in K562-CD19-GFP-Luc2 stable clone using the Bright-Lite Luciferase Detection System.

Figure 4: Characterization of CD19 overexpressing in K562-CD19-GFP-Luc2 stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI 1640 + 10% FBS + 1μg/mL Puromycin + 300μg/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 1:3-1:4 every 2 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. Francis J, Dharmadhikari AV, Sait SN, Deeb G, Wallace PK, Thompson JE, Wang ES, Wetzler M. CD19 expression in acute leukemia is not restricted to the cytogenetically aberrant populations. Leuk Lymphoma. 2013 Jul;54(7):1517-20. doi: 10.3109/10428194.2012.754096. Epub 2013 Jan 3. PMID: 23193950; PMCID: PMC6668030.
2. Cherian S, Miller V, McCullouch V, Dougherty K, Fromm JR, Wood BL. A novel flow cytometric assay for detection of residual disease in patients with B-lymphoblastic leukemia/lymphoma post anti-CD19 therapy. Cytometry B Clin Cytom. 2018 Jan;94(1):112-120. doi: 10.1002/cyto.b.21482. Epub 2016 Sep 23. PMID: 27598971.