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The cytokine concentrations were determined from standard curves, using murine recombinant cytokines as a standard.
Murine recombinant IL-17A for i.t.
Previously, we have shown that administration of murine recombinant TNF-α (rTNF-α) together with murine recombinant MCP-1 (rMCP-1) locally induced TNF-α release and psoriasiform skin lesions in mice [11].
The cytokine concentrations were determined with reference to a standard curve for serial twofold dilutions of the murine recombinant cytokines.
Cytokine levels were determined by comparison with standard curves generated from murine recombinant cytokines and analyzed using Bio-Plex Manager 4.0 software (Bio-Rad).
Murine recombinant PrP 23 231, used for indirect quantification of purified PrP aggregates, was expressed and purified according to the procedure of Zahn et al. [47] and stored in 50 mM sodium acetate, pH 4.0.
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Recombinant murine SCF, recombinant murine TNF-α and ICAM-1, purified anti-TNF-α and ICAM-1, and biotin-conjugated anti- TNF-α and ICAM-1 were purchased from R&D system (Minneapolis, MN, USA).
Mouse endothelioma (sEND-1) cells (8×105 per 35 mm well) were treated with 0 10 ng/mL murine-recombinant tumor necrosis factor alpha (TNF-α, R&D systems, UK) for 20 hours at 37°C to induce endothelial VCAM-1 expression.
Recombinant murine IL-2 and recombinant human IL-2 were administrated at a final concentration of 5 ng/mL (equivalent to 100 IU/mL).
After Matrigel polymerization, crypt culture medium (Advanced DMEM/F12 containing 50 ng/ml recombinant murine EGF, 1 μg/ml recombinant murine R-Spondin 1, 100 ng/ml recombinant murine Noggin, 1× N2 supplement, 1× B27 supplement, 1 mM N-acetylcysteine, 2 mM GlutaMax, 10 mM HEPES, and 100 U/ml penicillin/100 mg/ml streptomycin) was applied.
Recombinant murine IL-17A was purchased from Prospec (Rehovot, Israel) and recombinant murine IL-23 was purchased from R&D Systems.
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