Committed to providing comprehensive support for global drug development

The CD8 molecule, encoded by the CD8 gene, is a critical co-receptor on the surface of T cells. Primarily expressed on cytotoxic T lymphocytes (CTLs), it exists either as a heterodimer formed by disulfide-linked α (CD8α) and β (CD8β) subunits or as an α-α homodimer. Functioning as an accessory molecule to the T-cell receptor (TCR), CD8 binds to major histocompatibility complex class I (MHC I) molecules via its extracellular domain; this interaction enhances the sensitivity of the TCR to antigen-MHC complexes, thereby activating the cytotoxic function of T cells to precisely eliminate virus-infected or tumor cells.

The CD8 gene is located on human chromosome 2 (2p12) and comprises multiple exons, generating distinct mRNA isoforms through alternative splicing. Its expression is regulated by factors such as the transcription factor Runx3; it plays a pivotal role during the positive selection stage of thymic T-cell development, ensuring that T cells acquire the ability to recognize self-MHC molecules. Clinically, CD8+ T cells serve as core effector cells in tumor immunity; their infiltration density positively correlates with patient prognosis in various cancers (such as melanoma and colorectal cancer), and they are key targets for immune checkpoint inhibitors (e.g., anti-PD-1 therapies).

Figure 1. Recombinant Cyno CD8 CHO stably expressing Cyno CD8a & CD8b.

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 CD8 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.