Fluorescent labeling allows for imaging and tracking of vesicles down to singleparticle level. Among several options to introduce fluorescence, staining of lipid membranes with lipophilic dyes provides a straightforward approach without interfering with vesicle content. However, incorporating lipophilic molecules into vesicle membranes in an aqueous solution is generally not efficient because of their low water solubility. Here, we describe a simple, fast (<30 min), and highly effective procedure for fluorescent labeling of vesicles including natural extracellular vesicles. By adjusting the ionic strength of the staining buffer with NaCl, the aggregation status of DiI, a representative lipophilic tracer, can be controlled reversibly. Using cell-derived vesicles as a model system, we show that dispersion of DiI under low-salt condition improved its incorporation into vesicles by a factor of 290. In addition, increasing NaCl concentration after labeling induced free dye molecules to form aggregates, which can be filtered and thus effectively removed without ultracentrifugation. We consistently observed 6-to 85-fold increases in the labeled vesicle count across different types of dyes and vesicles. The method is expected to reduce the concern about off-target labeling resulting from the use of high concentrations of dyes.

Membrane-bound vesicles such as extracellular vesicles (EVs) can function as biochemical effectors on target cells. Docking of the vesicles onto recipient plasma membranes depends on their interaction with cell-surface proteins, but a generalizable technique that can quantitatively observe these vesicle-protein interactions (VPIs) is lacking. Here, we describe a fluorescence microscopy that measures VPIs between single vesicles and cell-surface proteins, either in a surface-tethered or in a membrane-embedded state. By employing cell-derived vesicles (CDVs) and intercellular adhesion molecule-1 (ICAM-1) as a model system, we found that integrin-driven VPIs exhibit distinct modes of affinity depending on vesicle origin. Controlling the surface density of proteins also revealed a strong support from a tetraspanin protein CD9, with a critical dependence on molecular proximity. An adsorption model accounting for multiple protein molecules was developed and captured the features of density-dependent cooperativity. We expect that VPI imaging will be a useful tool to dissect the molecular mechanisms of vesicle adhesion and uptake, and to guide the development of therapeutic vesicles. K E Y WO R D SCD9, cell-derived vesicles, ICAM-1, integrin, total internal reflection fluorescence microscopy, vesicleprotein interactions  INTRODUCTIONMembrane-bound vesicles can effectively elicit biochemical changes in cells. For example, extracellular vesicles (EVs), which are naturally secreted by virtually all types of cells, can adhere to the plasma membranes of target cells and trigger intracellular signalling, often accompanied by cargo transfer upon internalization. This process is therefore believed to enable effective Minkwon Cha and Sang Hyeok Jeong contributed equally to this work.

Several studies on the effect of water treatment sludge (WTS) have been conducted on trace metal contents of sorghum hybrid (Sorghum bicolor (L.) Moench) grown on a mountainous Kumsan district in the Republic of Korea. The objective of the present study was for measuring manganese (Mn) content in sorghum hybrid on an appropriate lamp current with an atomic absorption spectrophotometer. Four treatments were used in this experiment namely: a) without fertilizer (control), b) fermented alum sludge (Compost), c) alum sludge+nitrogen, phosphorus, potassium (Alum+NPK), and d) fermented alum sludge+nitrogen, phosphorus, potassium (Compost+NPK). After sorghum's harvest, the Mn content was investigated with a spectrophotometer on the conditions of three lamp currents; 4, 5, and 6 mA. Several ratios between the background and the absorbance values might be a very useful index for deciding the condition of the Mn analysis. The three ratios, (mean of backgrounds)/(standard deviation of absorbances), (standard deviation of backgrounds)/(Standard deviation of absorbances), and (2 x Standard deviation of backgrounds)/(mean of absorbances), were considered to be good tools for appropriate Mn analysis. As a result, 5 mA current was favorable for the analysis, therefore, the Mn content was calculated on 5 mA lamp current. There were significant differences of Mn contents among the four treatments: (Alum+NPK) < control, compost or (compost+NPK). The Mn contents of the sorghum for all the four treatments were higher than 40 mg/kg DM (dry matter), and they were considered over-optimum level for the ruminants' feed.

No abstract