Ch, Kyoto University, Uji, Japan; c NanoFCM Inc., Xiamen, China (People’s Republic); dDepartment of Chemical PDE11 manufacturer Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China, Xiamen, China (People’s Republic)aIntroduction: Lipoproteins co-isolate with EVs and are potential confounders in EV characterisation. CD36 is actually a membrane-bound scavenger receptor situated on cells and EVs capable of interacting with VLDL and LDL, which could interfere with antibody-based phenotyping. Freezing and thawing samples was shown to enhance phosphatidylserine-positive (PS+) EVs though other prevalent phenotype markers were unchanged. This could give a process for disrupting lipoproteins and EVs. Therefore, we aimed to investigate the effect of lipoproteins on EV characterisation and freezing/thawing samples on their dissociation from EVs on a high-resolution flow cytometer (hFCM). Approaches: Plasma from 6 healthy individuals was subjected to either 0, 2, 4 or 6 freeze-thaw (FT) cycles and stained with a cocktail of lactadherin-FITC, anti-CD41BV510, anti-CD36-PE and anti-ApoB-APC or lactadherin-FITC and matched isotype controls. Samples have been analysed on an Apogee A60 Micro-PLUS hFCM. Gating was performed as follows: size gates established on silica reference beads; phenotype gates set on 99th percentile of isotype control channel fluorescence. Final results: hFCM was capable to detect each free apolipoprotein B (ApoB) particles and ApoB bound to PS +CD41+, PS+CD36+ and PS+CD41+ CD36+ EVIntroduction: In all domains of life archaea, bacteria and eukarya, cells produce and release extracellular vesicles (EVs). The double-layered lipid membrane will be the most prominent feature of EVs, and fluorescent labelling with lipid-binding dyes has been often made use of to visualize and detect single EVs. By way of example, most standard flow cytometers rely on fluorescence threshold triggering for single EV detection upon membrane labelling with lipophilic dyes. However, the labelling efficiency of EVs with these lipid-binding dyes remains unknown. Right here, we reported an approach to quantitatively analyse the labelling efficiency of lipid-binding dyes toward EVs by using a laboratorybuilt nano-flow cytometer (nFCM) that enables light scattering detection of individual EVs as modest as 40 nm. Methods: EVs were extracted from cultured medium of HCT15 cells (colorectal cancer cell line), E. coli O157:ISEV2019 ABSTRACT BOOKH7 (gram-negative), S. aureus (gram-positive) and Prochlorococcus (Pro., marine cyanobacteria) by differential ultracentrifugation. EVs isolated from E. coli O157:H7 and S. aureus were additional purified by floatation in iodixanol density gradient. The purity of these EV isolates was assessed by enumerating the particles just before and immediately after the treatment with Triton X-100. Subsequently, the labelling efficiency of quite a few lipophilic fluorescent dyes, like PKH26, PKH67, DiI and Di-8-Ane for EVs have been evaluated by comparing with their light scattering signals. Results: The purity of EVs isolated from HCT15 cells, E. coli O157:H7, S. aureus and Pro. had been around 80 to 90 . Compared with side scattering NK1 list signals, we found that almost all of the EVs derived from E. coli O157:H7, S. aureus and Pro. could possibly be lightened up by PKH26, PKH67, DiI and Di-8-Anepps. Having said that, only around 40 of EVs isolated from HCT15 cells may be labelled by these dyes. Morphological study by cryoTEM indicates that some vesicles secreted by HCT15 cells had surface protrusions (electron-dense spi.