KC-6506

CTLL-2-PD1 Cell Line

62861

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Background of CTLL-2-PD1 Cell Line

PD-1, human programed cell death 1 protein, is a receptor belonging to immunoglobulin superfamily which is expressed primarily on activated T cells. NK cells, B cells, and monocytes. PD-1 interaction with its ligands, PD-L1 and PD-L2, leads to downregulation of T cell responses, including T cell proliferation and cytokine production, and limits immune-destruction of tissues. The interaction between PD-1 and its ligands, thus plays a role in maintaining the balance between immune activation and tolerance, potentially including tumor tolerance.

Specifications

Catalog Number	KC-6506
Cell Line Name	CTLL-2-PD1 Cell Line
NCBI/UniProt Accession Number	Q15116
Clone Number	6#
Host Cell Line	Mouse CTLL-2 Cell Line
Description	Stable CTLL-2 cell line expressing exogenous human PD1 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 +2μg/mL Puromycin+100ng/mL Mouse IL-2
Selection Marker	Puromycin
Morphology	Lymphoblast-like
Subculture	Split saturated culture 1:4-1:8 every 2-3 days
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 24 hours
Mycoplasma Status	Negative

Cell Line Generation

CTLL-2-PD1 cell line was generated using a lentiviral vector expressing the human PD1 sequence.

Characterization

Figure 1: Characterization of human PD1 overexpression in the CTLL-2-PD1 stable clone using FACS.

Figure 2: Characterization of human PD1 in the CTLL-2-PD1 stable clone using PCR sequencing.

Cell Resuscitation

Pre-warm complete culture medium (RPMI1640+10% FBS +2μg/mL Puromycin+100ng/mL Mouse IL-2) in a 37°C water bath.
Rapidly thaw the cryovial in a 37°C water bath for 1-2 minutes with gentle agitation.
Transfer the vial to a biosafety cabinet, and disinfect the exterior with 70% ethanol.
Aseptically transfer the cell suspension dropwise into a sterile centrifuge tube containing 9.0 mL of pre-warmed complete medium.
Centrifuge at approximately 125 × g for 5–7 minutes at room temperature, carefully aspirate the supernatant without disturbing the cell pellet.
Gently resuspend the pellet in an appropriate volume of complete medium and transfer the suspension into a T25 flask.
Incubate the flask in a 37°C in a humidified 5% CO₂ incubator.
Assess cell viability and morphology after 24 hours. If cells appear healthy, replace the medium with fresh medium supplemented with the appropriate selective antibiotic.
Subculture the cells at a ratio of 1:4-1:8 every 2-3 days upon reaching 80%–90% confluency.

Cell Freezing

Prepare the freezing medium (70% basal medium, 20% FBS and 10% DMSO) freshly before use.
Pre-chill the freezing medium on ice and label the cryovials accordingly.
Transfer the cell suspension to a sterile conical tube and perform a cell count to determine total viability and density.
Centrifuge the cells at 250×g for 5 minutes at room temperature; carefully aspirate the supernatant.
Gently resuspend the cell pellet in chilled freezing medium, ensuring a minimum cell density of 3×10⁶ cells/mL.
Aliquot 1 mL of the cell suspension into each pre-labeled cryovial.
Place the cryovials into a CoolCell® container and store at -80°C overnight for controlled-rate cooling.
Transfer the cryovials to the liquid nitrogen for long-term storage the following day.

References

1. Sunshine, J. & Taube, J. M. PD-1/PD-L1 inhibitors. Current Opinion in Pharmacology 23, 32–38 (2015).
2. Boussiotis, V. A. Molecular and Biochemical Aspects of the PD-1 Checkpoint Pathway. N Engl J Med 375, 1767–1778 (2016).
3. Topalian, S. L. et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366, 2443–2454 (2012).