Mip 1β

Mouse plasma samples were diluted 1:2 in assay buffer and analyzed via a multiplex bead analysis approach. Interleukin-1β (IL-1β), IL-2, IL-5, IL-17, IL-6, IL-4, IL-10, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), interferon-γ (IFNγ), tumor necrosis factor α (TNF-α), monocyte chemotactic protein-1 (MCP-1), regulated upon activation normal T cell expressed and secreted factor (RANTES), Eotaxin, Macrophage Inflammatory Protein-1β (MIP-1β), IL-23, and TNF-β were measured according to the manufacturer’s instructions (Millipore Corp., USA). All samples were analyzed on the Luminex 200^TM (Luminex, Austin, TX, USA) platform.

Plasma cytokine and chemokine concentrations were assayed in duplicate using a macrophage inflammatory protein 1α (MIP-1α) singleplex kit and a 22-analyte multiplex panel [granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma interferon (IFN-γ), interleukin-1 (IL-1) receptor agonist (IL-1RA), IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12/23(p40), IL-13, IL-15, IL-17, IL-18, monocyte chemoattractant protein 1 (MCP-1), MIP-1β, sCD40L, transforming growth factor α (TGF-α), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF)] (both from Millipore [catalog number PRCYTOMAG-40K]). The assays were performed according to the manufacturer’s recommended protocol, and the results were read using a FlexMAP three-dimensional array reader (Luminex Corp.). The data were analyzed using Bio-Plex Manager software (Bio-Rad).

The level of plasma cytokines was detected by Luminex assay. Plasma was centrifuged from peripheral blood samples and stored in −80 °C. The following 12 cytokines were measured with a Luminex kit following the manufacturer’s instructions: IFN-γ, IL-2, MCP-1, MIP-1β, IL-1β, IL-6, TNF-α, IL-8, IL-10 (Merck Millipore, Billerica, MA, USA, PRCYTOMAG-40K-09C), IFN-α (Carlsbad, CA, USA, EPX01A-40216-901), IP-10 (Carlsbad, CA, USA, EPX01A-40284-901), and TGF-β (Merck Millipore, Billerica, MA, USA, TGFBMAG-64K-01). Firstly, cryopreserved plasma samples were thawed and mixed completely. A total of 25 μL of plasma samples and 25 μL of assay buffer were added into the corresponding well of 96-well plates and mixed with 25 μL of magnetic beads. The plates wrapped with tinfoil were incubated with agitation for 16–18 h at 4 °C. After incubation, the plates were washed and incubated with 25 μL of detection antibody for 1 h at room temperature (RT). Finally, 25 μL of streptavidin-PE was added to plates and incubated for 30 min at RT. The plates were washed and infiltrated with 150 μL sheath fluid. Plates were loaded on a Luminex^® 200 (Bio-Rad, Hercules, CA, USA), and the results were analyzed for median fluorescent intensity using a five-parameter logistic method for calculating analyte concentration.

Multiplex analysis based on the xMAP Luminex technology was performed with the use of MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel—Premixed 38 Plex-Immunology Multiplex Assay (sCD40L, EGF, Eotaxin/CCL11, FGF-2, Flt-3 ligand, Fractalkine, G-CSF, GM-CSF, GRO, IFN-α2, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IP-10, MCP-1, MCP-3, MDC/CCL22, MIP-1α, MIP-1β, TGF-α, TNF-α, TNF-β, VEGF) (Merckmillipore, Burlington, MA, USA), in accordance with the manufacturer’s instructions. Briefly, samples were incubated with fluorescent beads for 1 h, washed and incubated with phycoerythrin-streptavidin for 10 min (Merckmillipore, Burlington, MA, USA). The analysis was done using a Luminex 200 analyzer (Merckmillipore, Burlington, MA, USA). The CIMVs-MSCs and MSCs lysates in IP buffer (50 mMTris-Cl, 150 mMNaCl, 1% Nonidet-P40) were used for multiplex analysis. Equal protein load (25 μg) was used for the analysis.

Blood samples were centrifuged for 10 min at 600 × g, and serum was immediately aliquoted and stored at −80°C. The following 11 cytokines were measured with a Luminex kit following the manufacturer’s instructions: IL-1β, IL-2, IL-6, IL-8, IL-10, IFN-γ, MCP-1, MIP-1β, TNF-α (Merck Millipore, Billerica, MA, United States, PRCYTOMAG-40K-09C), TGF-β (Merck Millipore, Billerica, MA, United States, TGFBMAG-64K-01), and IP-10 (Carlsbad, CA, United States, EPX01A-40284-901). After thawing the samples on ice and sufficient mixing, 25 μl of supernatant was loaded into each well of a 96-well plate and mixed with 25 μl of assay buffer and 25 μl of magnetic beads. The plates were incubated with agitation overnight at 4°C. After washing, 25 μl of detection antibody was added to each well, and the plate was incubated for 1 h at room temperature (RT). Then, 25 μl of streptavidin-PE was added to each well and incubated for 30 min at RT. Next, 150 μl sheath fluid was added to each well after washing. Plates were read on a Luminex^® 200 (Bio-Rad, Hercules, CA, United States), and the data were analyzed for median fluorescent intensity using a five-parameter logistic method for calculating analyte concentration.