Cells were monitored and quantified using a Multisizer 4 Coulter counter (Beckman Coulter, Nyon, Switzerland). For extracellular viral production, samples were filtered using 0.45 μM PVDF filters (Millex-HV, Millipore). Filtrate was fixed with a final concentration of 0.5% glutaraldehyde for 30 min at 4°C, then plunged into liquid nitrogen, and stored at -80°C until analysis. After thawing, 2:75 ratio of fixed sample was stained with SYBER gold (Invitrogen) prepared in Tris–EDTA buffer as instructed by the manufacturer (5 μl SYBER gold in 50 mL Tris–EDTA), then incubated for 20 min at 80°C and cooled down to room temperature. Flow cytometric analysis was performed with excitation at 488 nm and emission at 525 nm.
Calculation of infectious particles during infection (Fig C inS1 Text ) was done by using the most probable number (MPN) method as described in [20 (link)]. Briefly, medium was of infected culture was subjected to a series of fivefold dilutions for each sample. Each dilution (10 μl) was then added, in six technical replicates, to 100 μl of exponentially growing E. huxleyi cultures in multiwell plates over four or five days. MPN was calculated using the MPNcalc software.
Calculation of infectious particles during infection (Fig C in