Milliplex analysis software version 3

The level of inflammatory cytokines, tumor necrosis factor α (TNF-α), interleukin-4 (IL-4), IL-10, and IL-6 in pancreas, and lung and intestine tissue samples were assessed using the Milliplex MAP Rat Cytokine/Chemokine magnetic bead immunoassay kit (Millipore Corporation, Billerica, MA) following the manufacturers' instructions [29 (link), 30 (link)]. Briefly, diluted tissues homogenate samples and multiple premixed microbeads were added to a plate and the plate was incubated with agitation on a shaker. Then, the appropriate antibody detection mix was added into each well after washing. Each antibody was specific to a single cytokine. The plate was read on a MAGPIX Luminex xMAP instrument (Luminex Corp, Austin, TX) and analyzed with MILLIPLEX Analysis software version 3 (Millipore Corporation, Billerica, MA).

The level of secreted VEGF, IL-1β, IL-6, and IL-8 in the conditioned medium was quantified using MILLIPLEX MAP Human Cytokine/Chemokine Panels (Millipore) in a MAGPIX system (Luminex, Austin, TX) according to the manufacturer's protocol. Briefly, 25 µl of each sample was incubated with a mixture of magnetic beads coated with antibodies specific to each target cytokine/chemokine in a 96-well panel at 4°C overnight. The beads mixture with attached cytokine was washed twice and then incubated with detection antibody solution for 1 hr at room temperature. We then added 25 µl of the streptavidin-phycoerythrin solution to the panel and incubated for 30 min at room temperature. The beads were washed twice and re-suspended in the drive fluid and the panel was transferred into the MAGPIX instrument. The signal intensity of each cytokine/chemokine was then determined based on a parallel standard curve created in the panel using Luminex xMAP software (Luminex) and analyzed with MILLIPLEX Analysis software version 3 (Millipore).