Recombinant mouse il 18

Manufactured by BioLegend

Recombinant mouse IL-18 is a cytokine produced in a cell expression system. It is a member of the interleukin-1 family and plays a role in the immune response.

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5 protocols using recombinant mouse il 18

Cell Surface and Intracellular Staining

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The fluorescently conjugated antibodies which were used for cell-surface staining and intracellular staining are listed in the Supplementary Table 1. CD1d-PBS57 (conjugated with PE) was obtained from the tetramer facility of the US National Institutes of Health as previously described^{27 (link)}. Collagenase I used for tissue digestion was purchased from Gibco. Murine IL-2 and murine IL-12 p70 were obtained from PeproTech. Recombinant Mouse IL-18 was obtained from BioLegend.

Shen H., Gu C., Liang T., Liu H., Guo F, & Liu X. (2020). Unveiling the heterogeneity of NKT cells in the liver through single cell RNA sequencing. Scientific Reports, 10, 19453.

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Isolation and Activation of Mouse Splenocytes

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Splenocytes from wild-type and IL18RKO mice were isolated as described above. Splenocytes (10⁶ cells/mL) were cultured in 96-flat bottom well plates in RPMI (Gibco 21875), 1% Pen/Strep (BI 030311B), 10% FBS (Gibco 012657), 1 mM sodium pyruvate (BI 030421B), 10 mM Hepes (BI 030251B), and 10 μM β-mercaptoethanol (Gibco 31350–010). In addition, cells were cultured with or without CD3/CD28 beads (Miltenyi 130-095-925; 4:1 ratio cell to bead), and 10 ng/mL or 100 ng/mL recombinant mouse IL18 (Biolegend 767002). Cells were cultured at 37°C, 5% CO₂ for 48 or 96 hr. Afterward, cells were harvested and stained as described earlier to proceed with Treg sorting for RNA sequencing or CCR6 evaluation by flow cytometry.

Peligero-Cruz C., Givony T., Sebé-Pedrós A., Dobeš J., Kadouri N., Nevo S., Roncato F., Alon R., Goldfarb Y, & Abramson J. (2020). IL18 signaling promotes homing of mature Tregs into the thymus. eLife, 9, e58213.

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HDM Sensitization and Challenge Protocol

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For preparation of HDM immunizations, whole crushed D. pteronyssinus HDM powder (Greer Laboratories Inc.) was resuspended in sterile PBS. For experiments that performed allergic sensitization and challenge, mice were anesthetized with isofluorane and administered HDM via the oropharynx at a concentration containing 23 μg Der p 1 protein in a volume of 40 μl during the primary sensitization. Beginning at 10 d after sensitization, mice were anesthetized with isofluorane and instilled with HDM in the oropharynx at a concentration containing 5.75 μg Der p 1 protein daily for 5 d during the allergic challenge phase.
For acute respiratory challenge experiments with HDM, papain, LPS, and recombinant cytokines, mice were anesthetized with isofluorane and administered the target molecules in a total volume of 40 μl diluted in sterile PBS via the oropharynx for the indicated days. On each day, mice were given HDM normalized to 5.75 μg Der p 1 protein (Greer Laboratories Inc.), 25 μg papain (from papaya latex, aseptically filled; Sigma-Aldrich), 10 μg Ultrapure LPS from E. coli O111:B4 (InvivoGen), 50 ng recombinant mouse IL-33 (BioLegend), and/or 500 ng recombinant mouse IL-18 (BioLegend) as described in the relevant figures.

Rüterbusch M.J., Hondowicz B.D., Takehara K.K., Pruner K.B., Griffith T.S, & Pepper M. (2023). Allergen exposure functionally alters influenza-specific CD4+ Th1 memory cells in the lung. The Journal of Experimental Medicine, 220(11), e20230112.

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Cytokine-stimulated IFN-γ Production in NK Cells

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For cytokine stimulation experiments, 2 × 10 4 mouse NK cells were stimulated for 4 h in CR-10 (RPMI 1640 + 25 mM HEPES (Gibco-15630-080) + 10% FBS, 1% l-glutamine, 1% 200 mM sodium pyruvate, 1% MEM-NEAA, 1% penicillin-streptomycin, 0.5% sodium bicarbonate and 0.01% 55 mM 2-mercaptoethanol), brefeldin A (1:1,000 dilution; BioLegend) and monensin (2 μM; BioLegend) with or without recombinant mouse IL-15 (50 ng ml -1 ; PeproTech), mouse IL-12 (20 ng ml -1 ; PeproTech) and/or recombinant mouse IL-18 (10 ng ml -1 ; BioLegend, 767002). Cells were cultured in CR-10 medium alone as a negative control (no treatment). The absolute number of IFN-γ-producing NK cells were determined by acquiring and counting individual cells with an Attune NxT after intracellular flow cytometry staining to determine cell count of IFN-γ + of the 2 × 10 4 cells plated per condition. For plate-bound antibody stimulation experiments, 2 × 10 4 isolated NK cells for each condition were stimulated with 4 mg ml -1 precoated antibody against NK1.1 (PK136) for 4 h in complete medium containing brefeldin A (1:1,000 dilution; BioLegend) and monensin (2 μM; BioLegend). Cells were cultured in medium alone as a negative control (no treatment).

Cheng M.I., Li J.H., Riggan L., Chen B., Tafti R.Y., Chin S., Ma F., Pellegrini M., Hrncir H., Arnold A.P., O'Sullivan T.E, & Su M.A. (2023). The X-linked epigenetic regulator UTX controls NK cell-intrinsic sex differences. Nature immunology, 24(5).

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Cardiac Function Evaluation in IL-18-Treated Mice

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Male A/J mice (000646) were purchased from the Jackson Laboratory. Mice were housed in specific pathogen-free animal facilities at the Johns Hopkins University School of Medicine. Experiments were conducted with 8- to 12-week-old age-matched mice. Mice were intraperitoneally injected with 1 μg of recombinant mouse IL-18 (BioLegend) in 100 μL PBS every other day for 8 days. On day 10, cardiac function was monitored using M-mode transthoracic echocardiography, and then mice were sacrificed. To isolate single cells, hearts were perfused with PBS for 3 minutes, minced using razor blades, and incubated in 5 mL tissue digestion enzyme solution for 30 minutes at 37°C with agitation. The digestion enzyme solution was prepared with 600 U/ml Collagenase (Worthington), 100 U/ml DNase I (Worthington), and 100 U/ml Hyaluronidase (Sigma). Cells were washed and filtered through 40 μm cell strainers (Falcon).

Won T., Gilotra N.A., Wood M.K., Hughes D.M., Talor M.V., Lovell J., Milstone A.M., Steenbergen C, & Čiháková D. (2022). Increased Interleukin 18-Dependent Immune Responses Are Associated With Myopericarditis After COVID-19 mRNA Vaccination. Frontiers in Immunology, 13, 851620.

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