Hemolymph Cell Viability Assay

Aliquots (200 μl) of whole hemolymph (each containing about 1–2 × 10⁶ cells/ml) were incubated with the OlyA6/PlyB mixture (25 and 50 μg/ml) for 30 min at 16°C. Control samples were run in parallel. Total hemocyte count was carried out using an Omnicyt flow cytometer (Cytognos SL, Salamanca, Spain). Samples were pelleted by centrifugation (100 × g for 10 min) and stained with different fluorophores. Annexin V-FITC (ANX)/propidium iodide (PI) and TMRE staining was carried out as previously described (Ciacci et al., 2012 (link); Canesi et al., 2015 (link)); MitoSOX and CM-H2DCFDA staining was performed as described above for confocal scanning laser microscopy. Samples were also analysed by the Intracellular Glutathione Detection Assay Kit (Abcam) (at ex: 490, em: 520 nm). Sample acquisition and analyses were performed by means of a FACS Canto II flow cytometer and analysed with DiVa™ software collecting at least 10,000 events for each sample. Data, representing the mean ± standard deviation (SD) of at least three experiments, are expressed as Mean Fluorescence Intensities reported as percent changes with respect to controls, except for ANX/PI staining, where data are expressed as percent positive cells with respect to controls.