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Human Leukocyte Antigen G (HLA-G) is a non-classical Class I HLA molecule primarily expressed by extravillous trophoblasts in the placenta, where it mediates maternal-fetal immune tolerance during pregnancy. Although the expression of HLA-G is restricted in healthy tissues, pathological conditions can induce its expression. HLA-G expression has been observed in various cancers, including colorectal cancer, breast cancer, melanoma, and ovarian cancer. HLA-G plays a positive role in regulating innate and adaptive immune responses and promoting tolerance, while playing a detrimental role in inducing mechanisms of immune evasion. A common mechanism for evading immune surveillance involves the loss or downregulation of classical Class Ia HLA antigens, coupled with the *de novo* expression of non-classical Class Ib HLA antigens (such as HLA-E, -F, and -G).

The primary HLA-G transcript encodes seven distinct isoforms via alternative splicing: HLA-G1, -G2, -G3, and -G4 are membrane-bound forms, whereas HLA-G5, -G6, and -G7 are soluble isoforms. HLA-G1 and -G5 are the only subtypes capable of binding to β2-microglobulin (β2M). While β2M serves as an additional binding site for receptors, it has been demonstrated that the binding of HLA-G1 and -G5 to their receptors does not necessarily require the presence of bound β2M.

Serving as target cells for LILRB2 Effector Reporter Cells, B2M-free HLA-G aAPC Cells effectively mimic the *in vivo* signal transduction processes of LILRB2; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the B2M-free HLA-G aAPC Cell Model

Figure 2. Recombinant B2M-free HLA-G/aAPC Cell stably expressing HLA-G.

Figure 3. Dose Response of LILRB2 Blocking Antibody in LILRB2 Effector Reporter Cell (C24) with B2M-free HLA-G aAPC Cell.

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 Human B2M-free HLA-G/aAPC 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.