Chemidoc mp chemiluminescence imager

For western blotting detection, a total of 30 μg of T-EVs or crude protein extracted from cell lysates was separated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Danvers, Massachusetts, USA). The membranes were blocked with 5% BSA in TBST and then incubated with appropriate primary antibodies overnight at 4 °C. After incubation with horseradish peroxidase-coupled secondary antibodies for 1 h, the membranes were scanned using a ChemiDoc MP Chemiluminescence imager (Bio-Rad, Hercules, California, USA) according to the manufacturer's instructions. For FCM analysis, 20 μg of EVs was incubated with 5 μL of 4-μm-diameter aldehyde/sulfate latex beads (Invitrogen) for 15 min at room temperature in PBS, with a final volume of 20 μL. The mixture was then transferred to 1 mL of PBS and incubated with gentle shaking for 1 h. After centrifugation, the pellet was blocked by incubation with 20 μL of fetal bovine serum for 30 min. EV-coated beads were washed three times in PBS and resuspended in 50 μL of PBS. Afterward, the beads were incubated with appropriate fluorophore-conjugated antibodies for 1 h at room temperature in the dark. The beads were analyzed by FCM (Beckman Coulter) 38 (link).

For the large-scale phenotypic screening of transformants, 2 mL of a dense algal culture was concentrated on a 100 µm nylon screen and resuspended in 400 µL of assay buffer (0.1 M K2HPO4 pH 7.6, 0.5 M NaCl, 1 mM EDTA) [73 (link)]. The algae cells were then disrupted by 15 s of ultrasound sonication, using a Sonopuls HD2070 sonicator (Bandelin Electronic, Berlin, Germany) at 70% power, under cooling with ice. Based on the measured chlorophyll concentration of each sample, the samples were equalized for lysate concentration by adding assay buffer. In a black 96-well polystyrene plate (Thermo Fisher Scientific, Wilmington, DE, USA), 200 µL of each lysate was added to 50 µL of 10 µM coelenterazine (Fluka, Neu-Ulm, Germany) in assay buffer, using a multichannel pipette. Chemiluminescence was detected with a ChemiDoc MP chemiluminescence imager (Biorad, Hercules, CA, USA) and quantified using FIJI (ImageJ 1.51w) [78 (link)]. To document the uniformly adjusted lysate concentration of all samples based on their chlorophyll content, brightfield images were taken using a Nikon D200 camera.