KC-5774

Raji-OS8-PDL1 Cell Line

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64724

Background of Raji-OS8-PDL1 Cell Line

Raji-OS8 stable cell was used as an artificial antigen presenting cells (APCs), OS8 could function as a membrane anchored T cell engager that directly activates TCR in T cell-based assay.CD274, also commonly referred to as PDL1, This gene encodes 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. Expression of this gene in tumor cells is considered to be prognostic in many types of human malignancies, including colon cancer and renal cell carcinoma. Alternative splicing results in multiple transcript variants.

Specifications

Catalog NumberKC-5774
Cell Line NameRaji-OS8-PDL1 Cell Line
NCBI/UniProt Accession NumberNM_014143.4
Clone Number1#
Host Cell LineRaji
DescriptionRaji cell line stably expressing exogenous OS8 and PDL1 gene
QuantityTwo vials of frozen cells (≥2-106/vial)
StabilityStable in culture over a minimum of 10 passages
ApplicationDrug screening and biological assays
Freezing Medium70% RPMI1640 + 20% FBS + 10% DMSO
Propagation MediumRPMI1640+10%FBS+2µg/mL puromycin+400μg/mL Hygromycin B
Selection MarkerPuromycin | Hygromycin B
MorphologySuspension
SubcultureSplit saturated culture 1:4-1:6 every 2-3 days; seed out at about 1-3 × 105 cells/mL
Incubation37 °C with 5% CO2
StorageLiquid nitrogen immediately upon receiving
Doubling TimeApproximately 30 hours
Mycoplasma StatusNegative

Cell Line Generation

Raji-OS8-PDL1 cell line was generated using a lentiviral vector expressing the OS8 and PDL1 sequence.

Characterization

Figure1: Characterization of PDL1 overexpressing in Raji-OS8-PDL1 stable clones using FACS.

Cell Resuscitation

  1. Prewarm culture medium (RPMI1640+10%FBS+2µg/mL puromycin+400μg/mL Hygromycin B) 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 1:4-1:6 every 2-3 days; seed out at about 1-3 × 105 cells/mL.

Cell Freezing

  1. Prepare the freezing medium (70% RPMI1640 + 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. Akisada N, Nishimoto K, Takao S, Gion Y, Marunaka H, Tachibana T, Makino T, Miki K, Akagi Y, Tsumura M, Toji T, Yoshino T, Nishizaki K, Orita Y, Sato Y. PD-L1 expression in tongue squamous cell carcinoma. Med Mol Morphol. 2021 Mar;54(1):52-59. doi: 10.1007/s00795-020-00261-7. Epub 2020 Jul 22. Erratum in: Med Mol Morphol. 2021 Mar;54(1):68. doi: 10.1007/s00795-020-00270-6. PMID: 32699939.
  2. Koh J, Ock CY, Kim JW, Nam SK, Kwak Y, Yun S, Ahn SH, Park DJ, Kim HH, Kim WH, Lee HS. Clinicopathologic implications of immune classification by PD-L1 expression and CD8-positive tumor-infiltrating lymphocytes in stage II and III gastric cancer patients. Oncotarget. 2017 Apr 18;8(16):26356-26367. doi: 10.18632/oncotarget.15465. PMID: 28412752; PMCID: PMC5432263.

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.
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