Fluorescein isothiocyanate (fitc)

Fresh hemolymph and EPFs were carefully extracted as described before. Samples were filtered through a 100 μm nylon mesh, transferred into micro-tubes and kept on ice to minimize cell clumping, in a similar way to that described for hemolymph collection in Suminoe oyster [38 (link)]. Filtered samples were short centrifuged at 16,000 g, 13 seconds, 4°C. Cell pellets containing hemocytes were further washed with filtered clean sea water to remove debris. Flow cytometry assays were perfomed on both intact and fixed-permeabilized hemocytes. Acetone was selected as the Fixation-Permeabilization Solution (10 min at –20°C). Cell pellets were incubated overnight at 4°C with 100 μL of M22.8 hybridoma supernatant. To avoid non-specific binding, a blocking agent (FcR Blocking agent, Immunostep) was added for 10 minutes. Goat anti-mouse IgG (H+L) antibodies coupled to fluorescein isothiocyanate (FITC, Southern Biotech, USA) were used as secondary antibodies (30 min, 4°C). Unstained cells, i.e., samples not incubated or only incubated with secondary antibodies, were included as control cells. Samples were analyzed in a BD Accuri™ C6 Flow Cytometer (BD Biosciences) and the data were analyzed using BD Accuri C6 Software (eBiosciences).

Cells were stained with mouse anti-porcine CD4 monoclonal antibody, FITC (Southern Biotech, Birmingham, AL, USA) and Foxp3 monoclonal antibody, PE (eBioscience, San Diego, CA, USA) or with appropriate isotype controls. Attune NxT flow cytometer (Invitrogen, Carlsbad, CA, USA) and FlowJo software (Tree Star Inc., Stanford, CA, USA) were utilized for flow cytometric analysis.

A bead-based assay for detection of two classical exosome markers, CD63 and CD81 was used to phenotypically identify SEC fractions containing exosomes [26 (link)]. Briefly, exosomes were coupled with Aldehyde/Sulfate Latex Beads, 4% w/v, 4 µm (Invitrogen) and then blocked with PBS 1X/BSA 0.1% (Sigma-Aldrich) /NaN3 0.01% (Sigma-Aldrich). Fractions were incubated in microtest conical bottom 96-well plates for 30 min at 4 °C with anti-CD63 and anti-CD81 antibodies (culture supernatant monoclonal antibodies) at 1:10 dilution. After washing, a 1/100 dilution of secondary antibody FITC (Southern Biotech) was incubated for 30 min at 4 °C. After removal of unbound secondary antibodies by centrifugation, beads were suspended in PBS and analyzed by flow cytometry using aBD FACSVerse (BD Biosciences) equipment. Median Fluorescence Intensity (MFI) and beads count data were obtained by FlowJo analysis Software of every sample-reading file.