KC-2594

293T-LILRA5-Cell-Line

24370

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Background of 293T-LILRA5-Cell-Line

LILRA5, also known as CD85, encodes a protein that is a member of the leukocyte immunoglobulin like receptor (LIR) family. It is known that members of the LIR family have activation and initial functions in white blood cells. The cross-linking of this receptor protein on the surface of monocytes has been shown to induce calcium flux and the secretion of several pro-inflammatory cytokines, indicating the role of this protein in triggering innate immune responses. This gene is one of the white blood cell receptor genes that form gene clusters on chromosome region 19q13.4.

Specifications

Catalog Number	KC-2594
Cell Line Name	293T-LILRA5-Cell-Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous LILRA5 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% 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
In Vivo Validation	NA

Cell Line Generation

293T-LILRA5-cell-line was generated using a lentiviral vector expressing the LILRA5 sequence.

Characterization

Figure 1: Characterization of LILRA5 overexpression in the 293T-LILRA5 stable clone using FACS.

Figure 2: Characterization of endogenous LILRA5 expression in 293T cells using FACS.

Cell Resuscitation

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

Cell Freezing

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

References

Mitchell A, Rentero C, Endoh Y, Hsu K, Gaus K, Geczy C, McNeil HP, Borges L, Tedla N. LILRA5 is expressed by synovial tissue macrophages in rheumatoid arthritis, selectively induces pro-inflammatory cytokines and IL-10 and is regulated by TNF-alpha, IL-10 and IFN-gamma. Eur J Immunol. 2008 Dec;38(12):3459-73. doi: 10.1002/eji.200838415. PMID: 19009525.
Borges L, Kubin M, Kuhlman T. LIR9, an immunoglobulin-superfamily-activating receptor, is expressed as a transmembrane and as a secreted molecule. Blood. 2003 Feb 15;101(4):1484-6. doi: 10.1182/blood-2002-05-1432. Epub 2002 Sep 26. PMID: 12393390.
Shiroishi M, Kajikawa M, Kuroki K, Ose T, Kohda D, Maenaka K. Crystal structure of the human monocyte-activating receptor, Group 2 leukocyte Ig-like receptor A5 (LILRA5/LIR9/ILT11). J Biol Chem. 2006 Jul 14;281(28):19536-44. doi: 10.1074/jbc.M603076200. Epub 2006 May 3. PMID: 16675463.