Biotek powerwave ht microplate spectrophotometer

Exosome preparations were assessed by ELISA according to the manufacturer’s instructions. The following ELISA kits were used: mouse CCL2 DuoSet (R&D Systems, cat. #DY479); mouse IL-6 DuoSet (R&D Systems, cat. #DY40605); human CCL2 DuoSet (R&D Systems, cat. #DY279); human IL-6 DuoSet (R&D Systems, cat. #DY20605); human CD44 DuoSet (R&D Systems, cat. #DY704505); human glypican-1 DuoSet (R&D Systems, cat. #DY451905); human syndecan-1 ELISA Set (abcam, cat. #ab47352); human HSPG ELISA Kit (Perlecan) (abcam, cat. #ab274393); human versican ELISA Kit (Novus Biologicals, cat. #NBP275353). Absorbances were detected using a BioTeK PowerWave HT microplate spectrophotometer and the Gen5™ microplate reader software (BioTek, v2.09).

Exosomes (500 µg/ml) were incubated with increasing concentrations of either recombinant CCL2 (human: BioLegend, cat. #571406; mouse: R&D Systems, cat. #479JE-CF) or recombinant IL-6 (human: R&D Systems, cat. #206IL; mouse: Novus Biological, cat. #52156) cytokines in a final volume of 50 µl for 2 h at 37 °C under agitation. To remove free unbound cytokines, exosomes were re-purified using qEV columns (Izon Science Ltd), followed by sample concentration in Amicon Ultra-4 10-kDa nominal molecular weight centrifugal filter units (Merck Millipore) to a final volume of 200 µl. Cytokine binding was then assessed by ELISA. Absorbances were detected using a BioTeK PowerWave HT microplate spectrophotometer and the Gen5™ microplate reader software (BioTek, v2.09).
CCL2-conjugated exosome samples were also submitted to sonication (10 pulses of 5 s at 45% amplitude; 1-min intervals on ice) or treatment with 10 µg/ml of Proteinase K (Sigma-Aldrich, cat. #P2308) for 10 min at 37 °C before CCL2 detection by ELISA. Heat-inactivation of Proteinase K was performed by incubation at 90^oC for 5 min.