Streptomyces conglobatus

For FluoVolt experiment, 20 μL of packed RBCs were incubated with 1 mL of Hank's Balanced Salt Solution with calcium and magnesium (HBSS^+/+) and FluoVolt™ Membrane Potential Kit (Thermo Fisher) was added to the solution. RBCs were incubated at 37°C for 15 min in a rotating block. For generation of labelled RBC‐MVs, CellMask™ (Thermo Fisher) (1:1000 dilution) was added to 100 μL of packed RBCs diluted in 5 mL of HBSS^+/+. Cells were incubated at 37°C for 15 min in a rotating block. Then, Ionomycin 10 μM (Streptomyces conglobatus, I9657, Sigma‐Aldrich) or A23187 10 μM (C7522, Sigma‐Aldrich) were added to the cells and incubated at 37°C for 1 h in a rotating block. Cells were centrifuged twice at 2500 × g for 15 min (brakes off), and the supernatant was concentrated to 500 μL using a 10 kDa Amicon® (Millipore Sigma). RBCs‐MVs were purified by loading the concentrate into a qEV 35 nm size‐exclusion chromatography (SEC) column (Izon Science, Christchurch, New Zealand). Twenty 500 μL fractions were collected. For MVs analysis, the fractions 6–9 were pooled together and concentrated to 100 μL using a 100 kDa Amicon filter units.