Epx220 30122 901

After transcardial perfusion with 0.9 % NaCl, the dissected peri-infarct areas were lysed with ProcartaPlex cell lysis buffer (EPX-99999-000, Invitrogen) and homogenized with microbeads for 1 min. After centrifugation at 16,000 G for 10 min, total protein concentration of the supernatant was measured by BCA assay. Cytokine and chemokine 22-ProcartaPlex immunoassay (IL-1, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, G-CSF, GM-CSF, IFN, TNF, CXCL1, CXCL2, CXCL10, CCL2, CCL3, CCL5, CCL7 and CCL11) was performed according to manufacturer's recommendations with provided assay solutions (EPX220-30122-901, Thermo Fisher Scientific Inc.). Brief, magnetic beads were transferred into each well. After a washing step with a hand-held magnetic plate washer and wash buffer, universal assay buffer was added to each well and standards as well as samples (2.5 µg/µL of total protein) were added to the appropriate wells. After an overnight-incubation and washing steps, the detection antibody and streptavidin-PE were incubated for 30 min one after another with in-between washing steps. The samples were measured in reading buffer with Luminex Magpix Systems and Luminex xPonent Software (Merck Millipore).