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Members of the Leukocyte Immunoglobulin-like Receptor B (LILRB) family act as negative regulators of myeloid cell activation. LILRBs are Type I transmembrane glycoproteins that function as immunosuppressive receptors; currently identified members of the LILRB family include LILRB1, LILRB2, LILRB3, LILRB4, and LILRB5.

LILRBs are capable of broadly binding to MHC Class I molecules; subsequent studies revealed that their ligands also include members of the non-HLA Angiopoietin-like (ANGPTL) protein family, as well as S100A8, S100A9, APOE, and Bone Marrow Stromal Cell Antigen 2 (BST2). The ANGPTL family consists of glycoproteins primarily secreted by the liver; with the exception of ANGPTL8, all members of this family possess an N-terminal coiled-coil domain and a C-terminal fibrinogen-like domain. To date, eight ANGPTLs (ANGPTL1–ANGPTL8) have been identified, all of which function as angiopoietins to regulate angiogenesis.

The CHO-K1 Human ANGPTLx/TCR Activator Cell Model—effectively simulates the signal transduction process of LILRB4 *in vivo*. The underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the CHO-K1 Human ANGPTLx/TCR Activator Cell Model

Figure 2. Dose Response of LILRB4 Blocking Antibody in LILRB4 Effector Reporter Cell(C5) with ANGPTLx TCR Activitor CHO(C1).

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 ANGPTLx/TCR Activator 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.