KC-2148

CT26-PDL1-PVR-Cell-Line

23482

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Background of CT26-PDL1-PVR-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. PVR, abbreviated as poliovirus receptor, is a transmembrane glycoprotein belonging to immunoglobulin superfamily. CD155 is involved in the cellular poliovirus infection in primates, and intercellular adherent junctions between epithelial cells.

Specifications

Catalog Number	KC-2148
Cell Line Name	CT26-PDL1-PVR-Cell-Line
Host Cell Line	Mouse CT26 cell line
Description	Stable CT26 cell line expressing exogenous human PDL1 PVR 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+10%FBS+10µg/mL Puromycin
Selection Marker	Puromycin
Morphology	Fibroblast
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 24 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

CT26-PDL1-PVR cell line was generated using a lentiviral vector expressing the PDL1&PVR sequence.

Characterization

Cell Resuscitation

Prewarm culture medium (DMEM + 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 1:4-1:10 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

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).
Koike S, Horie H, Ise I, Okitsu A, Yoshida M, Iizuka N, Takeuchi K, Takegami T, Nomoto A (October 1990). "The poliovirus receptor protein is produced both as membrane-bound and secreted forms". EMBO J. 9 (10): 3217ÿ 24.
Mendelsohn CL, Wimmer E, Racaniello VR (1989). "Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily". Cell. 56 (5): 855ÿ 65.
Maier MK, Seth S, Czeloth N, et al. (2007). "The adhesion receptor CD155 determines the magnitude of humoral immune responses against orally ingested antigens". European Journal of Immunology. 37 (8): 2214ÿ25.