KC-4892

293T-TREM2-DAP12 Cell Line

52825

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Background of 293T-TREM2-DAP12 Cell Line

TERM2, full named as Triggering receptor expressed on myeloid cells 2, is a membrane protein which expressed primarily in immune cells across many different tissues, and form a receptor signaling complex with TYROBP, also named as DAP12, then mediates signaling and cell activation after binding serval ligands, it plays an important role in different inflammatory diseases, especially Alzheimer's disease, and is also believed as a promising target for cancer therapy.
DAP12 (also known as TYROBP and KARAP) is expressed by NK cells and myeloid cells and mediates signalling for numerous cell-surface receptors expressed by these cells.

Specifications

Catalog Number	KC-4892
Cell Line Name	293T-TREM2-DAP12 Cell Line
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous human TREM2 and DAP12 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	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-TREM2-DAP12 Cell Line was generated using a lentiviral vector expressing the human TREM2-DAP12 sequence.

Characterization

Figure 1: Characterization of TREM2 DAP12 overexpression in the 293T TREM2 DAP12 stable clone using FACS.

Figure 2: Characterization of TREM2 DAP12 in the 293T TREM2 DAP12 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. Gingras MC, Lapillonne H, Margolin JF (March 2002). TREM-1, MDL-1, and DAP12 expression is associated with a mature stage of myeloid development. Molecular Immunology. 38 (11): 817–24. doi:10.1016/S0161-5890(02)00004-4. PMID 11922939.
2. Bouchon A, Dietrich J, Colonna M (May 2000). Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes. Journal of Immunology. 164 (10): 4991–5. doi:10.4049/jimmunol.164.10.4991.
3. Turnbull, I., Colonna, M. Activating and inhibitory functions of DAP12. Nat Rev Immunol 7, 155–161 (2007). https://doi.org/10.1038/nri2014.