KC-2528

293T-LILRB2-isoform2 Cell Line

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Background of 293T-LILRB2-isoform2 Cell Line

LILRB2, also named as ILT4, CD85d, is member of the leukocyte immunoglobulin-like (LIR) family, and primarily expressed on monocytes, dendritic cells and neutrophils. Recent studies show LILRB2 can potentially act as a myeloid immune checkpoint by reprogramming tumor- associated myeloid cells and provoking antitumor immunity. LILRB2 has multiple alternatively spliced isoforms. Isoform 2 is one of the clearly defined protein-coding variants, which has been successfully expressed as a functional membrane protein and is used for drug screening and biological assays.

Specifications

Catalog Number	KC-2528
Cell Line Name	293T-LILRB2-isoform2 Cell Line
NCBI/UniProt Accession Number	NM_001080978.4
Clone Number	1#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous LILRB2 gene isoform 2
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 + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 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
In Vivo Validation	NA

Cell Line Generation

293T-LILRB2-isoform2 cell line was generated using a lentiviral vector expressing the LILRB2 gene isoform 2 sequence.

Characterization

Figure: Characterization of LILRB2 overexpression in the 293T-LILRB2-isoform2 stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 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 1:4-1:5 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. Cella M, Dohring C, Samaridis J, Dessing M, Brockhaus M, Lanzavecchia A, Colonna M (June 1997). A novel inhibitory receptor (ILT3) expressed on monocytes, macrophages, and dendritic cells involved in antigen processing. J Exp Med. 185 (10): 1743–51. doi:10.1084/jem.185.10.1743.
2. Samaridis J, Colonna M (April 1997). Cloning of novel immunoglobulin superfamily receptors expressed on human myeloid and lymphoid cells: structural evidence for new stimulatory and inhibitory pathways. Eur J Immunol. 27 (3): 660–5. doi:10.1002/eji.1830270313.
3. Fanger, N A; Cosman D; Peterson L; Braddy S C; Maliszewski C R; Borges L (November 1998). The MHC class I binding proteins LIR-1 and LIR-2 inhibit Fc receptor-mediated signaling in monocytes. Eur. J. Immunol. 28 (11): 3423–34