KC-1321

CHOK1-LAG3-Cell-Line

21923

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Background of CHOK1-LAG3-Cell-Line

Lymphocyte-activation gene 3(LAG3), also known as CD223, is a member of immunoglobulin superfamily, expressed on activated T cells, NK cells, B cells and DC cells. LAG3 can bind to MHC II molecules and induce maturation of DC cells, and cytokine secretion of cytotoxic T cells and NK cells. LAG3 is a promising drug target of cancer therapy and autoimmune disease, its therapeutic antibodies including BMS-986016 and GSK2831781 had already been used in the clinical trial.

Specifications

Catalog Number	KC-1321
Cell Line Name	CHOK1-LAG3-Cell-Line
Host Cell Line	Chinese hamster ovary CHO-K1 cell line
Description	Stable CHOK1 cell line expressing exogenous human LAG3 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+5µg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial-like
Subculture	Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-2 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 24 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

CHOK1 human LAG3 cell line was generated using a lentiviral vector expressing the human LAG3 sequence.

Characterization

Cell Resuscitation

Prewarm culture medium (F12K + 10% FBS + 5µg/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 1-2 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% F12K + 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

Anderson, Ana C, Nicole Joller, and Vijay K Kuchroo. 2016. ¡ùLag-3, Tim-3, and TIGIT: Co-Inhibitory Receptors with Specialized Functions in Immune Regulation.¡ì Immunity 44 (5). Elsevier Inc.: 989Ã¿1004.
Huard, B, R Mastrangeli, P Prigent, D Bruniquel, S Donini, N El-Tayar, B Maigret, M Dr¹Èano, and F Triebel. 1997. ¡ùCharacterization of the Major Histocompatibility Complex Class II Binding Site on LAG-3 Protein..¡ì
Proceedings of the National Academy of Sciences 94 (11): 5744Ã¿49.