CircuLex Human ISG15 ELISA Kit [#CY-8085, Nordic Biosite]; human IFNγ [#88-7316-86, Invitrogen, Thermo Fisher Scientific, Bremen, Germany]; TNF [#DY210], CXCL10 [#DY266], CCL20 [#DY360] and CXCL8 [#DY208]; all [R&D]; were used according to the manufacturers’ protocol. Cytokines in conditioned medium from stimulated PBMCs were detected and analysed by Bio-Plex Pro™ Human Chemokine Panel, 40-Plex [#171ak99mr2, Bio-Rad laboratories] according to the manufacturer’s protocol. The Limulus Amebocyte Lysate [LAL]-assay [#50-647U, LONZA Walkersville, MD USA, QCL-100TM] was performed according to the manufacturer’s instructions to confirm that the levels of endotoxin were well below 1 EU [<0.1 ng] per µg protein, as stated by the suppliers of both recombinant IL12 and ISG15. Nevertheless, since even 0.02 ng/ml LPS has been shown to significantly stimulate small amounts of cytokine production,28 (link) we defined cytokine release to be modulated by ISG15 only if the enhanced cytokine concentration in conditioned medium was above 1000 pg/ml compared with untreated control in the multiplex assay.
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Quantifying Cytokine Modulation by ISG15
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Corresponding Organization : St Olav's University Hospital
Other organizations : Nordland Hospital, UiT The Arctic University of Norway, Oslo University Hospital, University of Oslo
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Variable analysis
independent variables
- ISG15
- Cytokine concentrations in conditioned medium from stimulated PBMCs (IFNγ, TNF, CXCL10, CCL20, CXCL8)
- Endotoxin levels (Limulus Amebocyte Lysate [LAL]-assay performed to confirm levels of endotoxin were well below 1 EU [<0.1 ng] per µg protein)
- Positive control: Not specified
- Negative control: Untreated PBMCs
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