KC-1906

PC3-PDL1-Trop2-Cell-Line

23027

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Background of PC3-PDL1-Trop2-Cell-Line

PD-L1, also called human programmed cell death ligand 1, is a transmembrane protein that plays 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 the cancer cell to evade the host immune system. Trop2, also named as epithelial glycoprotein 1 antigen (EGP-1), is a member of a family including at least two type I membrane protein, Trop2 acts as a cell surface receptor and transduce intracellular calcium signal, and is also a carcinoma associated antigen, which was the target of several therapeutic antibodies, including GA773, and sacituzumab govitecan.

Specifications

Catalog Number	KC-1906
Cell Line Name	PC3-PDL1-Trop2-Cell-Line
Host Cell Line	Human PC3 cell line
Description	PC3 cell line stable expressing exogenous human PDL1 and TROP2 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% F12K+20% FBS+10% DMSO
Propagation Medium	Ham's F12K+10%FBS+1µg/mL Puromycin+100µg/mL Hygromycin B
Selection Marker	Puromycin, HygromycinB
Morphology	Epithelial
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

PC3 Human PDL1-TROP2 Cell Line was generated using a lentiviral plasmid with human TROP2 gene sequence.

Characterization

Cell Resuscitation

Prewarm culture medium (Ham's F12K + 10% FBS + 1µg/mL Puromycin + 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 1:4-1:8 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

Sunshine, J. & Taube, J. M. PD-1/PD-L1 inhibitors. Current Opinion in Pharmacology 23, 32ÿ38 (2015).
Boussiotis, V. A. Molecular and Biochemical Aspects of the PD-1 Checkpoint Pathway. N Engl J Med 375, 1767ÿ 1778 (2016).
Topalian, S. L. et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366, 2443ÿ2454 (2012).
Linnenbach AJ, Wojcierowski J, Wu SA, et al. (1989). "Sequence investigation of the major gastrointestinal tumor-associated antigen gene family, GA733". Proc. Natl. Acad. Sci. U.S.A. 86 (1): 27ÿ31.
Fornaro M, Dell'Arciprete R, Stella M, et al. (1995). "Cloning of the gene encoding Trop-2, a cell-surface glycoprotein expressed by human carcinomas". Int. J. Cancer. 62 (5): 610ÿ8.
El Sewedy T, Fornaro M, Alberti S (1998). "Cloning of the murine TROP2 gene: conservation of a PIP2-binding sequence in the cytoplasmic domain of TROP-2". Int. J. Cancer. 75 (2): 324ÿ30.