KC-0984-DW

MC38-mouse-PDL1-KO-PDL1 Cell Line

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Background of MC38-mouse-PDL1-KO-PDL1 Cell Line

PD-L1, also called human programed cell death ligand 1, is a transmembrane protein that play a major role in suppressing the immune system during particular events such as pregnancy, tissue allografts, autoimmune disease, virus infection and cancer. PD-L1 binds to its receptor PD-1 on activated T cells, B cells, and myeloid cells, to modulate activation or inhibition. Upregulation of PD-L1 can allow cancer cell to evade the host immune system.

Specifications

Catalog Number	KC-0984-DW
Cell Line Name	MC38-mouse-PDL1-KO-PDL1 Cell Line
NCBI/UniProt Accession Number	NM_014143.4
Clone Number	NA
Host Cell Line	MC38
Description	Stable MC38-mouse-PDL1-KO cell line expressing exogenous PDL1 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% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+15%FBS+0.01mM NEAA+100μg/mL Hygromycin B
Selection Marker	Hygromycin B
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:10 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 30 hours
Mycoplasma Status	Negative

Cell Line Generation

MC38-mouse-PDL1-KO-PDL1 cell line was generated using lentivirus expressing PDL1 sequence.

Characterization

Figure 1. Characterization of mouse-PDL1 knockout and PDL1 over-expression in the MC38-mouse-PDL1-KO-PDL1 stable clone using FACS.

Figure 2:Validation of in vivo tumorigenicity of MC38-mouse-PDL1-KO cells stably expressing PDL1 via subcutaneous implantation in C57BL/6J mice, with tumor growth monitored by measuring volume. (V=0.5×length×width²)

Figure 3. Characterization of PDL1 over-expression in the MC38-mouse-PDL1-KO-PDL1 stable clone using PCR.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 15% FBS, 0.01mM NEAA+100μg/mL Hygromycin B)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 1:4-1:10 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 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

Akinleye, Akintunde, Yamei Chen, Nikhil Mukhi, Yongping Song, and Delong Liu. 2013. “Ibrutinib and Novel BTK Inhibitors in Clinical Development.” Journal of Hematology & Oncology 6 (1). Journal of Hematology & Oncology: 1–1. doi:10.1186/1756-8722-6-59.
Cheng, S, A Guo, P Lu, J Ma, M Coleman, and Y L Wang. 2014. “Functional Characterization of BTKC481S Mutation That Confers Ibrutinib Resistance: Exploration of Alternative Kinase Inhibitors,” September. Nature Publishing Group, 1–25. doi:10.1038/leu.2014.263.