Committed to providing comprehensive support for global drug development

The CD96 gene is located on human chromosome 3 (3q13.11) and encodes the CD96 molecule, a single-chain transmembrane glycoprotein belonging to the immunoglobulin superfamily. Its structure comprises three immunoglobulin-like domains (two V-type and one C-type); the extracellular segment undergoes N-glycosylation, the transmembrane region consists of 21 amino acids, and the intracellular segment contains 45 amino acids and binds to SH2 domain-containing proteins to participate in signal transduction. The primary function of CD96 is to act as a co-inhibitory receptor on natural killer (NK) cells and T cells; by binding to its ligand CD155 (PVR), it regulates immune cell activation and effector functions. This interaction inhibits NK cell cytotoxicity and cytokine secretion, playing a pivotal role in maintaining immune homeostasis.

Figure 1. Recombinant HEK293 Human CD96 Cell constitutively expressing CD96.

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 CD96 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.