KC-3077

CHOK1-KLRG1 Cell Line

34094

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Background of CHOK1-KLRG1 Cell Line

KLRG1 is a co-suppressor receptor that is expressed on NK cells and antigen-experienced T cells. KLRG1+ NK cells may play an anti-fibrotic role in the natural course of chronic hepatitis B infection. Utilizing this anti-fibrotic function may provide a new therapeutic approach for the treatment of liver fibrosis in patients with chronic hepatitis B.

Specifications

Catalog Number	KC-3077
Cell Line Name	CHOK1-KLRG1 Cell Line
Clone Number	NA
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous KLRG1 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
In Vivo Validation	NA

Cell Line Generation

CHOK1-KLRG1 Cell Line was generated using a lentiviral vector expressing the KLRG1 sequence.

Characterization

Figure 1: Characterization of KLRG1 overexpression in the CHOK1-KLRG1 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640+10%FBS+10μg/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% 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

Henson SM, Akbar AN. KLRG1--more than a marker for T cell senescence. Age (Dordr). 2009 Dec;31(4):285-91. doi: 10.1007/s11357-009-9100-9. PMID: 19479342; PMCID: PMC2813054.
Herndler-Brandstetter D, Ishigame H, Shinnakasu R, Plajer V, Stecher C, Zhao J, Lietzenmayer M, Kroehling L, Takumi A, Kometani K, Inoue T, Kluger Y, Kaech SM, Kurosaki T, Okada T, Flavell RA. KLRG1+ Effector CD8+ T Cells Lose KLRG1, Differentiate into All Memory T Cell Lineages, and Convey Enhanced Protective Immunity. Immunity. 2018 Apr 17;48(4):716-729.e8. doi: 10.1016/j.immuni.2018.03.015. Epub 2018 Apr 3. PMID: 29625895; PMCID: PMC6465538.
Ager CR, Zhang M, Chaimowitz M, Bansal S, Tagore S, Obradovic A, Jugler C, Rogava M, Melms JC, McCann P, Spina C, Drake CG, Dallos MC, Izar B. KLRG1 marks tumor-infiltrating CD4 T cell subsets associated with tumor progression and immunotherapy response. J Immunother Cancer. 2023 Sep;11(9):e006782. doi: 10.1136/jitc-2023-006782. PMID: 37657842; PMCID: PMC10476134.