NTA was performed using NanoSight LM10 instrument (Malvern Panalytical, Malvern, UK) with 488?nm laser and NTA3.1 software. in a high-throughput manner. We demonstrate that data obtained by luminescent quantification are well correlated with data obtained by standard nanoparticle tracking analysis under multiple conditions. In addition, our system is usually capable of evaluating the recipient cells or tissues that take up exosomes, as well as visualizing exosomes gene with Nluc using the CRISPR/Cas9 genome-editing system. To place the Nluc gene sequence upstream of the 3 terminal quit codon, we constructed a targeting vector and knock-in donor vector, and co-transfected both vectors into HCT116 cells (Fig.?4a). We selected some candidate clones by using luciferase activity as an indication of Nluc knock-in, and obtained CD63Nluc knock-in (KI) cells (clone#17) after confirming the introduction of Nluc by PCR (Supplementary Fig.?3). Finally, we sequenced the gene in this clone and confirmed homozygotic Nluc insertion at the preterminal position (Supplementary Fig.?3). Expression of Nluc-labeled CD63 was detected in whole cells and isolated exosomes only in CD63Nluc-KI #17 cells (Fig.?4b). Nluc knock-in did not show significant effects around the Boc-NH-PEG2-C2-amido-C4-acid localization of CD63 and the number TRIM13 and size of exosomes (Supplementary Fig.?3). As explained above for CD63Nluc-expressing cells, we analyzed the relationship between reporter signal intensity and cell number or exosome number in the culture medium. Reporter signals in the culture medium were closely correlated with both cell and exosome figures (Fig.?4c,d). Moreover, the curve depicting the correlation between luminescence and exosome number was linear in a statistically significant manner at concentrations above 106 particles/mL (Fig.?4e). Furthermore, to verify the reliability of CD63Nluc-KI #17 for exosome quantification, we monitored the alterations of exosome number and luminescence in the culture medium from cells treated with ALIX shRNA, bafilomycin A1, and Boc-NH-PEG2-C2-amido-C4-acid hypoxia. Under all conditions, changes in the luminescence of the culture medium reflected the alterations in the exosome number (Fig.?4f). Taken together, these results suggest that knock-in of Nluc into CD63 provides a useful tool for quantifying exosomes. Open Boc-NH-PEG2-C2-amido-C4-acid in a separate window Physique 4 Generation of CD63Nluc-knock-in-HCT116 cells. (a) Schematic representation for generating CD63Nluc knock-in-HCT116 cells. (b) Western blot analysis of Nluc-labeled intrinsic CD63 expression in cells (left panels) and purified exosomes (right panels). ALIX was used as an exosomal marker protein. (c) Correlation between luciferase intensity (in the culture medium) and cell number. The solid collection shows the linearity of the fitted curve between luminescence and Boc-NH-PEG2-C2-amido-C4-acid seeded cell number. (d) Correlation between luciferase intensity (in the culture medium) and exosome number. Solid collection shows the linearity of the fitted curve of luminescence vs. exosome number. (e) Detection limits of CD63Nluc-KI#17-HCT116 cells for exosome quantification. Purified exosomes were adjusted to a concentration of 1010 particles/mL, and then a dilution series was prepared down to a concentration of 106 particles/mL. Detection limits were determined by comparing luciferase intensities of the dilution series with those of buffer (20?mM HEPES, pH7.4). (f) Alteration of exosome number (upper panels) and luminescence (lower panels) in the culture medium following treatment of CD63Nluc-KI#17-HCT116 cells with ALIX shRNA (left panels), bafilomycin A1 (middle panels), or hypoxia (1% O2) (right panels). Results are expressed as means??SD of three wells. All data are representative of at least three-independent experiments. **imaging of cells, proteins, and molecules such as drugs. Therefore, we investigated whether cells secreting CD63Nluc-labeled exosomes are useful for analyzing the biodistribution of exosomes. Exosomes secreted from cells constantly circulate throughout the whole body via the blood. Therefore, we developed an experimental system that persistently releases exosomes luciferase (Gluc), from your marine copepod luciferase (36?kDa)27. These properties of Nluc make it the most suitable luciferase for labeling of exosomes. Therefore, we developed a cell-based exosome quantification system using Nluc. Compared to the UC-NTA method, Boc-NH-PEG2-C2-amido-C4-acid our Nluc-based exosome measurement system has two main disadvantages:.