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NK cell receptors (NKRs) are expressed on the surface of NK cells and T cells, participating in the regulation of downstream signaling activation and inhibition, and actively involved in the regulation of immune responses. They possess a C-type lectin-like domain, which loses its ability to bind Ca2+, thus participating in protein-protein interactions rather than carbohydrate binding.

KLRB1 protein (NKR-P1A or CD161) CD161 (KLRB1) is a type II transmembrane protein of the C-type lectin-like receptor, expressed on NK cells and CD4+ and CD8+ T cell subsets. As an inhibitory receptor for mature NK cells, it inhibits NK cell cytotoxicity and cytokine secretion. Blocking or inactivating CD161 can enhance the killing effect of T cells against gliomas, which is beneficial for controlling tumor growth in vivo.

Lectin-like transcript 1 (LLT1 or CLEC2D) is a ligand for the CD161 receptor expressed on natural killer cells and T cells. CD161 inhibits NK cell cytotoxicity and IFN-γ secretion by binding to LLT1 expressed on activated leukocytes. The LLT1/CD161 interaction simultaneously shuts down NK cell activation while co-stimulating T cells.

Figure 1. Recombinant Cyno CD161(Medium Expression) CHO constitutively expressing Cyno CD161.

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  CHO-K1 Cynomolgus CD161(Medium Expression) 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.