Cy7 mono nhs ester

In vivo imaging experiments were performed as previously described [71 (link)]. EV fraction (15 µg) was incubated with 5 µM Cy7 Mono NHS Ester (GE Healthcare, Buckinghamshire, UK) for 90 min at 37 °C. The unincorporated dye was removed using Exosome Spin Columns (MW. 3000) (ThermoFisher). Animal experiments were performed in compliance with the guidelines of the Ethics Committee of Animal Care and Experimentation of Tokushima University (approval number T29-31). Eight-week-old female BALB/c mice (CLEA, Tokyo, Japan) were administered with 15 µg Cy7-labeled EVs per mouse by intraperitoneal injection. At 30 min after the injection, Cy7 fluorescence in the various organs of mice was analyzed by the IVIS Spectrum imaging system (Caliper Life Sciences, A PerkinElmer Company, Hopkinton, MA, USA).

LpEVs (µg) were incubated with 5 μM Cy7 mono NHS ester (GE Healthcare, Little Chalfont, UK) for 1 h at 37 °C. Cy7 mono NHS ester-labeled LpEVs were isolated using ultracentrifugation. Then, Cy7-labeled LpEVs (10 μg of total protein) were administered by gavage to the mice, which had been fasted overnight. At the indicated time point, whole-body images were obtained at a wavelength of 780–800 nm using a Davinch-Invivo system (Davinch-Invivo Fluoro Chemi, Korea). After whole-body imaging, the mice were sacrificed, and Cy7 fluorescence in the dissected organs was quantified.

Bacteria or bacterial EVs were incubated with 5 μM Cy7 mono NHS ester (GE Healthcare, Little Chalfont, UK) for 1 h at 37 °C. Cy7 mono NHS ester-labbeled samples were isolated using ultracentrifugation. Then, bacteria or bacterial EVs (20 μg of total protein) were administered by gavage to each mouse that had been fasted overnight. At the indicated time point, whole body images were obtained at 780–800 nm wavelength using IVIS® spectrum CT (SelectScience, Wilmslow, UK). After 12 h, the mice were sacrificed and Cy7 fluorescence was quantified in tissues including the liver, fat, and muscle.

After incubation of ^MousefEVs with Cy7-mono-NHS ester (5 μM, GE Healthcare Life Sciences, Pittsburgh, PA, United States) for 2 h at 37°C, Cy7-labeled ^MousefEVs (^MousefEVs_Cy7) were isolated by centrifugation at 150,000 × g for 3 h at 4°C, as previously reported (Jang et al., 2015 (link)). Wild-type mice were intraperitoneally administered with ^MousefEVs_Cy7 (100 μg in total protein amounts). Before administration, as well as at 10 min, 3, 6, 12, and 24 h after administration, Cy7 fluorescence in the mice was measured by IVIS spectrum (Caliper Life Sciences, Hopkinton, MA, United States) at Pohang Center for Evaluation of Biomaterials, Pohang Technopark, Pohang, South Korea. At 3 and 24 h after administration, mice were sacrificed, and Cy7 fluorescence was quantified in organs including the liver, lung, spleen, kidney, heart, and small intestine. Living Image 3.1 software (Perkin Elmer, Waltham, MA, United States) was used to measure radiant efficiency, and the efficiency was normalized by tissue weights.