KC-5616

293T-KIR3DL3-Cell-Line

60004

Home » 293T-KIR3DL3-Cell-Line

Background of 293T-KIR3DL3-Cell-Line

KIR3DL3 is a killer cell immunoglobulin-like receptor located within the leukocyte receptor complex region on human chromosome 19. KIR3DL3 is highly expressed on naïve NK cells that have not encountered antigens. KIR3DL3 is one of the oldest members of the KIR gene family and is considered to be the ancestor from which other KIR genes evolved. Studying it helps to understand the evolutionary history of the entire KIR family. KIR genes exhibit high polymorphism and diversity. Specific alleles or haplotypes of KIR3DL3 have been studied for their association with susceptibility or resistance to certain diseases, including autoimmune diseases, infectious diseases (such as HIV and hepatitis B), and pregnancy complications.

Specifications

Catalog Number	KC-5616
Cell Line Name	293T-KIR3DL3-Cell-Line
NCBI/UniProt Accession Number	NM_153443.4
Clone Number	9#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous KIR3DL3 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

Cell Line Generation

293T-KIR3DL3-cell-line was generated using a lentiviral vector expressing the KIR3DL3 sequence.

Characterization

Figure 1: Characterization of KIR3DL3 overexpression in the 293T-KIR3DL3 stable clone using FACS.

Figure 2: Characterization of KIR3DL3 in the 293T -KIR3DL3 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.Bhatt RS, Berjis A, Konge JC, Mahoney KM, Klee AN, Freeman SS, Chen CH, Jegede OA, Catalano PJ, Pignon JC, Sticco-Ivins M, Zhu B, Hua P, Soden J, Zhu J, McDermott DF, Arulanandam AR, Signoretti S, Freeman GJ. KIR3DL3 Is an Inhibitory Receptor for HHLA2 that Mediates an Alternative Immunoinhibitory Pathway to PD1. Cancer Immunol Res. 2021 Feb;9(2):156-169. doi: 10.1158/2326-6066.CIR-20-0315. Epub 2020 Nov 23. PMID: 33229411; PMCID: PMC8284010.
2.Campbell KS. Mystery Checkpoint Revealed: KIR3DL3 Finally Found a Ligand in HHLA2. Cancer Immunol Res. 2021 Feb;9(2):128. doi: 10.1158/2326-6066.CIR-20-0996. PMID: 33536266.
3.Palmer WH, Leaton LA, Campos Codo A, Crute B, Roest J, Zhu S, Petersen J, Tobin RP, Hume PS, Stone M, van Bokhoven A, Gerich ME, McCarter MD, Zhu Y, Janssen WJ, Vivian JP, Trowsdale J, Getahun A, Rossjohn J, Cambier J, Loh L, Norman PJ. Polymorphic KIR3DL3 expression modulates tissue-resident and innate-like T cells. Sci Immunol. 2023 Jun 30;8(84):eade5343. doi: 10.1126/sciimmunol.ade5343. Epub 2023 Jun 30. PMID: 37390222; PMCID: PMC10360443.