Human il 6 elisaready set go kit

Human PBMCs were purchased from Astarte Biologics (Redmond, WA), and the donors were negative for HIV-1 and -2, HBV, HCV, and HTLV. PBMCs were isolated from healthy donors’ whole blood according to the previously reported procedure51 (link). These studies on the use of human blood and tissue samples were performed and approved in accordance with the regulation of the Institutional Use Committee and Animal Care Committee of Korea Institute of Science and Technology (Ref. No. KIST 2016-043). Throughout the current studies, the informed consent was obtained from all subjects. We seeded the freshly isolated PBMC cells into 96-well plates at density of 2 × 10⁵ cells/well in RPMI 1640 supplemented with 10% FBS plus 2 mM L-glutamine. After the cells were completely attached, we stimulated them with various reagents as follows: monomeric siRNA (50 nM MDR1 siRNA plus 50 nM BCL2 siRNA), monomeric siRNA/PEI (branched, 25 kDa) (equivalent to 50 nM MDR1 siRNA plus 50 nM BCL2 siRNA), Dsi RNA transcripts (20 μg/mL), Dsi RNPs (26 μg/mL), and CpG ODN (5 μM) as a positive control of INF-α or resiquimod R-848 (10 μg/mL) as a positive control of IL-6. At 24 h post-treatment, we measured the released INF-α and IL-6 on the supernatants by using VeriKine™ Human INF-α ELISA Kit (PBL Biomedical) and Human IL-6 ELISA Ready-SET-Go Kit (eBioscience) following the manufacturer’s instruction.

Cytokines were measured in EVs isolated from 5 × 10⁵ human monocytes, 5 × 10⁵ human M1 macrophages, 1 × 10⁶ mouse monocytes, or 1 × 10⁷ human/mouse whole-blood cells. TGF-β1 was measured with Human TGF-β1 DuoSet ELISA (R&D Systems, cat no. DY240) and mouse TGF-β1 DuoSet ELISA (R&D Systems, cat no. DY1679) or Human/Mouse TGF-β1 ELISA Ready-SET-Go! Kit (eBioscience, cat no. 88-8350-88). IL-10, IL-6, and Il-1β were measured using the human IL-10 ELISA Ready-SET-Go! Kit (eBioscience, cat no. 88-7106-22), human IL-6 ELISA Ready-SET-Go! Kit (eBioscience, cat no. 88-7066-22), or human IL-1β ELISA Ready-SET-Go! Kit (eBioscience, cat no. 88-7261-86). In all cases, the ELISA was performed according to the manufacturer’s protocol. CD9 and HSP90 were measured with ELISA on immobilized vesicle using anti-CD9 antibody (Novus Biologicals, cat no. NB500-327) (2 µg/mL) and anti-HSP90 antibody (Abcam, cat no ab79848) (2 µg/mL), respectively. Vesicles in different fractions of size-exclusion chromatography was immobilized with TGF-β1 capture antibody from Human TGF-β1 DuoSet ELISA, and CD9 or HSP90 was detected on vesicles by biotinylated CD9 and HSP90 antibody and streptavidin-conjugated HRP.