Nightowl lb981 nc100 system

Ethical approval for this study was obtained from the Institutional Animal Care and Use Committee of Hoshi University (approval no. P21-039). A total of 8 female BALB/c mice (weight, 18–20 g; age, 8 weeks; Sankyo Labo Service Corporation, Inc.) were housed at 24°C and 55% humidity under 12/12-h light/dark cycle (lights on at 8:00 a.m.) with food and water ad libitum. siRNA lipoplexes with 20 µg Cy5-siRNA were administered intravenously to mice via the lateral tail vein (n=1/siRNA lipoplex). At 1 h post-injection of siRNA lipoplexes, mice were sacrificed via cervical dislocation; death was confirmed by cessation of heartbeat. Tissue (lung, heart, liver, spleen, and kidney) was analyzed by Cy5 fluorescence imaging using NightOWL LB981 NC100 system (Berthold Technologies GmbH & Co. KG), as previously described (21 (link)). The images were analyzed using IndiGo2 software (version 2.0.1.0) provided with the in vivo imaging system (Berthold Technologies). Following fluorescence imaging, tissue samples were frozen on dry ice and sliced into 16 µm sections. The localization of Cy5-siRNA was examined using a fluorescent microscope (Eclipse TS100-F; Nikon Corporation) with optical filter Cy5 HQ (excitation, 620/60 nm; dichroic mirror, 660 nm; emission, 700/75 nm; Nikon Corporation).

For efficient delivery of siRNA into liver metastasis, lipoplexes with 50 µg Cy5.5-siRNA were administered intravenously into mice with HeLa-liver metastasis at 1 min after the intravenous injection of 1 mg chondroitin sulfate (Wako Pure Chemical Industries, Ltd.) at 10 days after inoculation of HeLa-Luc cells, as reported previously (sequential injection method) (15 (link),16 (link)). A total of 1 h after injection, the mice were sacrificed, and Cy5.5 fluorescent imaging of the tissues was performed using a NightOWL LB981 NC100 system (Berthold Technologies, Bad Wildbad, Germany). In Cy5.5 fluorescent imaging, the excitation and emission filters were set at 630/20 and 680/30 nm, respectively. The exposure time for fluorescence was 5 sec. A grayscale body-surface reference image was collected using a NightOWL LB981 CCD camera (Berthold Technologies). The images were analyzed using IndiGo2 software (version 2.0.1.0; Berthold Technologies) provided with the in vivo imaging system.

All animal experiments were conducted in accordance with ‘Guide for the Care and Use of Laboratory Animals’ published by the U.S. National Institutes of Health and ‘Guide for the Care and Use of Laboratory Animals’ adopted by the Institutional Animal Care and Use Committee of Hoshi University (Tokyo, Japan), which is accredited by the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The Institutional Animal Care and Use Committee of Hoshi University approved this study (permission no. 20-018).
Seven female BALB/c mice (18–20 g, 8 weeks of age; Sankyo Labo Service Corp. Inc.) were housed at 24°C and 55% humidity with a 12/12 h light/dark cycle (lights on at 8:00 a.m.) and access to food and water ad libitum. Lipoplexes carrying 20 µg of Cy5.5-siRNA were injected into the lateral tail veins of mice (n=1 per siRNA lipoplex), then all mice were sacrificed by cervical dislocation 1 h later as described (19 (link)), and cervical dislocation was confirmed by careful assessment of the mice for explicit signs of death such as cardiac arrest. Tissues were analyzed by Cy5.5 fluorescence imaging using a NightOWL LB981 NC100 system (Berthold Technologies GmbH & Co. KG., Bad Wildbad, Germany) as described (14 (link)).