KC-2454

MC38-LAG3-Cell-Line

24094

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Background of MC38-LAG3-Cell-Line

LAG3, also known as CD223, is an immune checkpoint receptor protein primarily expressed in activated T cells NK cells B cells and plasma cell dendritic cells. LAG3 can downregulate T cell activity by binding to MHC II molecules. Meanwhile, LAG3 can also enhance the inhibitory activity of regulatory T cells (Tregs). The use of therapeutic antibodies to inhibit LAG3 can relieve the inhibition of T cells and enhance the body's immune response. LAG3 is also a new therapeutic target for various malignant tumors.

Specifications

Catalog Number	KC-2454
Cell Line Name	MC38-LAG3-Cell-Line
Host Cell Line	MC38
Description	Stable MC38 clone expressing exogenous 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% 1640 + 20% FBS + 10% DMSO
Propagation Medium	1640 + 10% FBS + 5μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:8-1:10 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 20 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

MC38-LAG3-cell-line was generated using a lentiviral vector expressing the LAG3 sequence.

Characterization

Figure 1: Characterization of LAG3 overexpression in the MC38-LAG3 stable clone using FACS.

Figure 2: Characterization of endogenous LAG3 expression in MC38 cells using FACS.

Cell Resuscitation

Prewarm culture medium (1640 supplemented with 10% FBS and 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 1:8-1:10 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% 1640 + 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

Zaitsu S, Yano M, Adachi S, Miwa M, Katoh T, Kawano Y, Yasuda M. Lymphocyte-activation gene 3 protein expression in tumor-infiltrating lymphocytes is associated with a poor prognosis of ovarian clear cell carcinoma. J Ovarian Res. 2023 May 13;16(1):93. doi: 10.1186/s13048-023-01179-1. PMID: 37179337; PMCID: PMC10182671.
1. Pedersen JM, Hansen AS, Skejø C, Juul-Madsen K, Junker P, Hørslev-Petersen K, Hetland ML, Stengaard-Pedersen K, Østergaard M, Møller BK, Dreyer L, Hauge EM, Hvid M, Greisen S, Deleuran B. Lymphocyte activation gene 3 is increased and affects cytokine production in rheumatoid arthritis. Arthritis Res Ther. 2023 Jun 7;25(1):97. doi: 10.1186/s13075-023-03073-z. PMID: 37287025; PMCID: PMC10246404.
Adashek JJ, Kato S, Nishizaki D, Miyashita H, De P, Lee S, Pabla S, Nesline M, Conroy JM, DePietro P, Lippman S, Kurzrock R. LAG-3 transcriptomic expression patterns across malignancies: Implications for precision immunotherapeutics. Cancer Med. 2023 Jun;12(12):13155-13166. doi: 10.1002/cam4.6000. Epub 2023 May 3. PMID: 37132280; PMCID: PMC10315766.