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Interleukin-12 (IL-12) is a heterodimeric pro-inflammatory cytokine that modulates T cell and natural killer cell responses, induces the production of interferon-γ (IFN-γ), and promotes helper T cell 1 (TH1) cells; it serves as a crucial link between innate and adaptive immunity. The IL-12 family also includes IL-23, IL-27, IL-35, and IL-39. IL-12 and IL-23 are produced by antigen-presenting cells (dendritic cells and tissue-resident macrophages) in humans and mice in response to exogenous or endogenous signals associated with host defense and wound healing, and are essential for the recruitment and effector functions of CD8 T and NK cells. IL-12 can neutralize the signaling of negative regulatory receptors on CD8 T cells. For example, IL-12 downregulates PD-1 and IFN-γ expression on CD8 T cells and protects tumor-infiltrating CD8 T cells from IFN-γ-induced cell death. Anti-PD-1 activation of antitumor immunity requires IL-12-mediated crosstalk between T cells and dendritic cells, thereby enabling CD8 T cell-mediated killing of tumor cells.

      IL-12 consists of an IL-12p35 subunit linked to an IL-12p40 subunit; signaling is mediated by a heterodimer composed of the IL-12 receptor (IL-12R), which contains IL-12Rβ1 and IL-12Rβ2 subunits. IL-12 stimulates JAK2 and TYK2 activity, leading to the phosphorylation of STAT1, STAT3, STAT4, and STAT5, members of the signal transduction and transcription activator (STAT) family.

 The IL-12 Effector Reporter Cell model effectively mimics the in vivo signaling pathway of IL-12; the mechanism is illustrated in the figure below.

Figure 1. Schematic diagram of the IL-12 Effector Reporter Cell model

Figure 2. Dose Response of Recombinant Human IL12 Protein in  IL12 Effector Reporter Cell(C2).

Figure 3. Inhibition of rhIL12-induced Reporter Activity by IL-12/IL -23 p40 Neutralizing Antibody in IL12 Effector Reporter Cell(C2).

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 IL12 Effector Reporter 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.