KC-5093

293T-B7H3-KO-mouse-PDL1-Cell-Line

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Background of 293T-B7H3-KO-mouse-PDL1-Cell-Line

PDL1 encoded by this gene is an immune inhibitory receptor ligand that is expressed by hematopoietic and non-hematopoietic cells, such as T cells and B cells and various types of tumor cells. The encoded protein is a type I transmembrane protein that has immunoglobulin V-like and C-like domains. Interaction of this ligand with its receptor inhibits T-cell activation and cytokine production. During infection or inflammation of normal tissue, this interaction is important for preventing autoimmunity by maintaining homeostasis of the immune response. In tumor microenvironments, this interaction provides an immune escape for tumor cells through cytotoxic T-cell inactivation. Mice deficient for this gene display a variety of phenotypes including decreased allogeneic fetal survival rates and severe experimental autoimmune encephalomyelitis.

Specifications

Catalog Number	KC-5093
Cell Line Name	293T-B7H3-KO-mouse-PDL1-Cell-Line
NCBI/UniProt Accession Number	NM_021893.3
Clone Number	8-2#
Host Cell Line	293T-B7H3-KO
Description	Stable 293T-B7H3-KO clone expressing exogenous mouse-PDL1 gene
Quantity	One vial 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 + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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

293T-B7H3-KO-mouse-PDL1-cell-line was generated using a lentiviral vector expressing the mouse-PDL1 sequence.

Characterization

Figure 1: Characterization of mouse-PDL1 overexpression in the 293T-B7H3-KO-mouse-PDL1 stable clone using FACS.

Figure 2:Characterization of mouse-PDL1 overexpression in the 293T-B7H3-KO stable clone using FACS.

Figure 3:Characterization of mouse-PDL1 in the 293T-B7H3-KO-mouse-PDL1 stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/mL puromycin)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1. Kuai L, Xiang YW, Chen QL, Ru Y, Yin SY, Li W, Jiang JS, Luo Y, Song JK, Lu B, Luo Y, Li B. PD-L1 Triggered by Binding eIF3I Contributes to the Amelioration of Diabetes-Associated Wound Healing Defects by Regulating IRS4. J Invest Dermatol. 2022 Jan;142(1):220-231.e8. doi: 10.1016/j.jid.2021.06.028. Epub 2021 Jul 20. PMID: 34293353.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.