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The *CD160* gene is located on human chromosome 1q21.1 and encodes a 27 kDa glycosylphosphatidylinositol (GPI)-anchored protein belonging to the immunoglobulin superfamily. Structurally, it comprises two extracellular immunoglobulin-like domains and is anchored to the cell membrane surface via a GPI anchor, lacking transmembrane or intracellular signaling domains.

Functionally, CD160 is primarily expressed on natural killer (NK) cells, NKT cells, intraepithelial T cells, γδ TCR+ T cells, as well as memory-phenotype, activated, and effector CD8+ T cells. It stimulates the activation of NK cells and CD8+ T cells through weak binding to MHC class I molecules and serves as a marker for cytolytic or exhausted CD8+ T cells. Furthermore, CD160 participates in co-inhibitory signaling; upon binding to HVEM (herpesvirus entry mediator), it inhibits CD4+ T-cell proliferation and TCR-mediated signaling, thereby exerting a bidirectional regulatory role in the immune response.

Figure 1. Recombinant HEK293 Human CD160 Cell stably expressing CD160.

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed HEK293 Human CD160 Cell complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.