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T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) is a cell-surface molecule containing both immunoglobulin and mucin domains. Originally identified as a cell-surface marker for IFN-γ-producing CD4 T helper 1 (Th1) and CD8 cytotoxic 1 (Tc1) cells, it serves as a critical immune checkpoint receptor. TIM-3 is primarily expressed on activated CD8+ T cells, exhausted T cells, Th1 cells, and innate immune cells (such as dendritic cells and macrophages). The TIM protein family consists of Type I transmembrane proteins sharing a similar structural architecture: a variable immunoglobulin (IgV) domain, a glycosylated mucin domain of varying length, and a single transmembrane domain. With the exception of TIM-4, all TIM molecules possess a C-terminal cytoplasmic tail containing conserved tyrosine-based signaling motifs. 

The current model of TIM-3 signaling posits that upon T-cell activation, TIM-3 is recruited to the immunological synapse, where Bat3 binds to the cytoplasmic tail of TIM-3 and recruits the catalytically active form of lymphocyte-specific protein tyrosine kinase (Lck). However, when TIM-3 engages with its ligand, conserved tyrosine residues within the cytoplasmic tail undergo phosphorylation; this event triggers the dissociation of Bat3, thereby enabling TIM-3 to exert its inhibitory function.

The TIM-3 Effector Reporter Cell model accurately recapitulates the in vivo signaling pathway of TIM-3; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the TIM-3 Effector Reporter Cell Model

Figure 2. Dose Response of TIM-3 Antibody in TIM-3 Effector Reporter Cell(C16) with TIM-3 Target Cell.

Cell Passage Procedures

1.This cell line grows in suspension.
2.Upon receipt, cells should be thawed immediately or stored in liquid nitrogen until use.
3.Before thawing, pre-warm the water bath and culture medium to 37 °C, and prepare a small amount of dry ice.
4.Remove the cryovial from storage and transport it to the cell culture laboratory on dry ice.
5.Rapidly thaw the cells in a 37 °C water bath. Once the cells are completely thawed, spray the cryovial with 70% ethanol for disinfection and transfer it to a biosafety cabinet.
6.Add 10 mL of pre-warmed culture medium into a 15 mL centrifuge tube. Transfer the contents of the cryovial into the tube and centrifuge at 1000 rpm for 5 minutes.
7.Carefully discard the supernatant. Resuspend the cell pellet in 5 mL of pre-warmed culture medium by gentle pipetting. Immediately perform cell counting and adjust the cell density to 3–6 × 10⁵ cells/mL based on the counting results, then transfer the cells into a culture flask.
8.Count the cells every 1–2 days. When the cell density exceeds 1 × 10⁶ cells/mL, passage the cells promptly or add fresh culture medium. Maintain the cell density between 2 × 10⁵ and 1 × 10⁶ cells/mL.

Suspension Cell Cryopreservation Procedure:

1.Collect 8 × 10⁶ cells, centrifuge, and discard the supernatant.
2.Add 1 mL of cell freezing medium (90% FBS + 10% DMSO) and gently pipette to mix thoroughly. Transfer the suspension into a cryovial.
3.Immediately place the cryovial into a controlled-rate freezing container (Nalgene 5100-0001), fill with isopropanol up to the indicated level, and store at −80 °C.
4.After 24 hours, transfer the cryovial to liquid nitrogen for long-term storage.