Imagestream x mark 2

WSSV virions were labeled with FITC (Sigma-Aldrich; 1.24546). The rMindin or tag, together with FITC-WSSV (1 × 10⁶ copies/shrimp), was injected into shrimp hemocoels. The hemolymph was drawn 1 h later into cold anticoagulant (0.45 M NaCl, 10 mM KCl, 10 mM EDTA, and 10 mM HEPES, pH 7.45) and centrifuged at 800 × g for 10 min at 4°C to obtain hemocytes. One part of the hemocytes was resuspended in PBS and detected using flow cytometry (ImageStreamX Mark II; Merck, Kenilworth, NJ, USA). The other part was placed onto poly-l-lysine-coated glass slides and washed three times with PBS. The nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI; AnaSpec, San Jose, CA, USA; AS-83210) for 10 min at room temperature. The slides were observed using an Olympus SpinSR10 confocal microscope (Olympus, Tokyo, Japan). The percentage of virus-positive cells was determined by counting the cells. Four visual fields were selected, and the number of cells in each visual field was not less than 100.

Phagocytosis assay was performed as described previously [53 ]. Briefly, isolated monocytes were stained with PKH67 (Sigma-Aldrich) according to the manufacturer's instructions and differentiated to macrophages by 20 ng/ml Macrophage-Colony Stimulator Factor (M-CSF) (R&D Systems) in X-VIVO 10 medium (Lonza) supplemented with 10% autologous serum. MOLM-13 cells were stained with PKH26 (Sigma-Aldrich) following the manufacturer's instructions and incubated in a 1:2 E:T ratio with licMABs or mAb concentrations ranging from 0.01 nM to 100 nM for 2 h. Polybead^® Carboxylate Red-Dyed Microspheres of 6 μm (Polysciences) were used as a positive control and incubation either at 4°C or at 37°C in the presence of 10 μM Cytochlasin D (Sigma-Aldrich) served as a negative control. Cells were harvested, measured by imaging flow cytometry using an ImageStream^®X Mark II instrument (Merck Millipore, Billerica, Massachusetts, USA) and analyzed with IDEAS^® and INSPIRE^® Software (Merck Millipore, Billerica, Massachusetts, USA). The maximum phagocytosis value was set to 100% and all data points were normalized accordingly. Mean values and standard errors of triplicates were calculated and plotted.

MB488⁺ sEVs were stained with antibodies for 30 minutes at RT, resuspended in PBS up to 200 μL, filtered (0.22 μm), and incubated at 4°C overnight before acquisition with the ImageStreamX Mark II imaging flow cytometer (EMD Millipore). Stained MB488⁺ sEVs were acquired by setting the imaging flow cytometer as previously published (57 ). Briefly, the instrument was set to low-speed/high-sensitivity mode (60× magnification), and the power for the used lasers was set to the maximum.