kc-6296

Jurkat-CD59-KO Cell Line

60963

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Background of Jurkat-CD59-KO Cell Line

The CD59 gene, located on chromosome 11p13, encodes a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein that serves as a pivotal regulator of the complement system. CD59, also known as MAC-inhibitory protein (MAC-IP) or protectin, functions by binding to the C8 and C9 components of the nascent membrane attack complex (MAC), thereby inhibiting its final assembly and pore formation on host cell membranes. This activity is essential for protecting "self" cells from unintended complement-mediated lysis, a process critical for maintaining tissue homeostasis and preventing autologous damage. The role of CD59 extends beyond basic immune regulation. Its expression on a wide variety of cells, including erythrocytes, endothelial cells, and immune cells, highlights its systemic importance in complement restriction. Dysregulation or deficiency of CD59 is implicated in several pathological conditions. For instance, in paroxysmal nocturnal hemoglobinuria (PNH), a acquired deficiency of GPI-anchored proteins like CD59 on blood cells leads to chronic complement-mediated hemolysis. Furthermore, many cancer types, such as glioblastoma and colorectal carcinoma, frequently overexpress CD59 as a key mechanism of complement evasion, contributing to tumor survival, progression, and resistance to antibody-based therapies. Consequently, CD59 has emerged as a significant therapeutic target, with strategies ranging from blocking antibodies to CAR-T cells designed to overcome this immunosuppressive barrier in the tumor microenvironment.

Specifications

Catalog Number	kc-6296
Cell Line Name	Jurkat-CD59-KO Cell Line
NCBI/UniProt Accession Number	966/P13987
Clone Number	3B3
Host Cell Line	Jurkat
Description	Stable Jurkat clone with CD59 gene knockout
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	RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS
Selection Marker	NA
Morphology	lymphoblast
Subculture	Split saturated culture 1:4-1:6 every 2-3 days; seed out at about 1 × 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

Jurkat-CD59-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of Jurkat-CD59-KO cell line stable clone using PCR sequencing.

Figure 2: Characterization of Jurkat-CD59-KO Cell Line stable clone using RT-PCR sequencing.

Figure 3: Characterization of Jurkat-CD59-KO Cell Line stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS) 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 1:4-1:6 every 2-3 days; seed out at about 1 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Bubeck D, Pardo SA, Giddings K, Girdler E, Deme JC, Johnson S, Le Bas A, Morgan BP, Lea SM. Structural basis for membrane attack complex inhibition by CD59. Nat Commun. 2023 Feb 1;14(1):573. doi: 10.1038/s41467-023-36441-z. PMID: 36725856; PMCID: PMC9892082.
Lv Z, Bian Z, Shi L, Niu S, Ha B, Tremblay JR, Ghosh S, Xu C, Zhao Y, Gildener-Leapman N, Ohm JE, Wang S, Zhang Z, Li Q, Liu R. Silencing EGFR-upregulated expression of CD55 and CD59 activates the complement system and sensitizes lung cancer to checkpoint blockade. Nat Cancer. 2022 Oct;3(10):1192-1210. doi: 10.1038/s43018-022-00443-5. Epub 2022 Oct 17. PMID: 36253609.