Ivis lumina 3 in vivo imaging system

Experiments were carried out using 8–12 weeks old mice under the protocol approved by IACUC. Mice were injected intraperitoneally with either vehicle control (15% HS15 in normal saline) or WY14643 (100 mg/kg) for 1 week starting from 3 days before virus infection. Mice were then infected with MHV68-M3FL at the dose of 10⁶ PFU through intraperitoneal route (31 (link), 34 (link)). To quantify virus-encoded luciferase expression, mice were weighed and injected with 150 mg/kg of d-Luciferin (GOLDBIO) immediately prior to imaging using an IVIS Lumina III In Vivo Imaging System (PerkinElmer). A region of interest (ROI) was drawn around the abdominal region and applied to all mice. Total flux (photons/second) of the ROI was measured (exposure = 1 sec., F/stop = 1.2, FOV=E, binning=medium, emissions filter=open) using Living Image software (PerkinElmer). Survival of the mice was recorded until 20 days after infection.

For the dual-luciferase assay, the promoter constructions inserted into pGreenII 0800-LUC were used in the analysis. The A. tumefaciens GV3101 strains harboring the promoter were cultured at 28 °C overnight. The resuspension buffer (10 mM MgCl₂, 10 mM MES, and 100 mM acetosyringone) was used to dilute the cultures to an OD600 of 0.6. The pFeUGT2::LUC, pFtUGT2::LUC, pFeS-RNase::LUC, and pFtS-Rnase::LUC were injected into separate N. benthamiana leaves and then cultured 2 days in the dark and 1 day in the light at 25 °C. The injected leaves were then detached and sprayed with 1 mM D-Luciferin sodium salt (Solarbio Beijing) + 0.01%Triton X-100. The luciferase luminescence from the infiltrated area was imaged using the IVIS Lumina III In Vivo Imaging System (PerKinElmer, Germany).

For the xenograft studies, 6-week-old female BALB/c nude mice were purchased from SLAC Laboratory Animal Co., Ltd (Shanghai, China) and maintained under pathogen-free conditions. A total of 4.5 × 10⁶ Lv-miR-340-A2780 or control cells in 100 µL PBS were subcutaneously (s.c.) injected into the rear flank of the mice. Tumors were measured using a caliper, and tumor volume was calculated using the following formula: V = L × W² × 0.5236 (L = long axis, W = short axis). For in vivo metastasis assays, 1 × 10⁶ Lv-miR-340-SKOV3 or 2 × 10⁶ Lv-miR-340-A2780 cells and the corresponding control cells in 200 µL PBS were injected intraperitoneally (i.p.) into the nude mice. After 70 days, the mice were photographed, and then euthanized. The nodules throughout the peritoneal cavity were counted and the ascites weight was measured. The intestines were subjected to fluorescent image detection using the IVIS Lumina III In Vivo Imaging System (PerkinElmer, MA, USA). All animals were maintained and treated in accordance with the guidelines of the Institutional Animal Care and Use Committee of Wenzhou Medical University.

Cytokine assays were performed using a Human Neuro Discovery Array C2 (RayBiotech, Norcross, GA, USA) according to the manufacturer’s instructions. Membranes precoated with cytokine antibodies were blocked with a blocking buffer at room temperature for 30 min; next, equal volumes of supernatants, collected from HMC3 cells treated, with or without 1 µg/ml LPS in the presence or absence of hADSCs or EVs, were added to replace the blocking buffer and incubated overnight at 4 °C. After washing three times with wash buffer 1 and two times with wash buffer 2, each membrane was incubated with biotin-conjugated antibodies for 2 h followed by a HRP-conjugated streptavidin at room temperature for 30 min. Membranes were developed by Detection buffer C and D mixture and the chemiluminescence was visualized by an IVIS Lumina-III In Vivo Imaging System (PerkinElmer). Densitometry analysis of the array was performed using the NIH Image J software.

8–12 weeks old BALB/c mice were lethally irradiated with 800 cGy split in two doses separated by 3 h. 4 h after irradiation, mice were transplanted with 5 × 10⁶ BM cells, 2 × 10⁶ splenocytes, depleted from monocytes, and 3 × 10⁵ freshly purified Tregs from C57BL/6 donors, and 10⁶ A20 leukemic cells transduced with a GFP-Luciferase vector^{64 (link)}, depending on the treatment group. Ruxolitinib (30 mg/kg day) or vehicle was administered via oral gavage from day + 1 until the end of the experiment. Luminescence was measured at days + 7 and + 13 using a IVIS Lumina III in vivo imaging system (PerkinElmer, Massachusetts, USA) as previously described^{64 (link)}.

Female nude mice were obtained from Jackson Laboratory. Experimental protocols were approved by the Institutional Animal Care and Use Committee at Wake Forest health Science. Female NU/NU Nude Mice from Charles River Laboratories were used for animal experiments. Mice were housed in a 12h light/12 h dark cycle with temperature-controlled room. The animal rooms are provided with 100% fresh, HEPA filtered air at 10-15 air changes per hour. Room temperatures are controlled by reheat units within each room, and are maintained within the range of 70°F ± 2° F. The humidity levels are controlled globally, and it is maintained between 30-70%. The mice were fed with a standard chow (Prolab Isopro RMH 3000, 5P00, LabDiet) and water ad libitum. Approximately 1 million cells were subcutaneously injected into the mammary fat pad of 7- to 9-week-old mice. Tumor length (L) and width (W) were measured by caliper, and tumor size was calculated using the formula, 1/2* LW^{2 (link)}. The maximum tumor size is 2cm of the length and maximal tumour size/burden was not exceeded in this study. Tumor growth were also monitored by bioluminescence imaging using the IVIS lumina III in vivo imaging system (Perkin Elmer). Living Image (Caliper Life Science) version 4.7.3 was used to analyze the bioluminescence level. Tumor wet weight were measured at the endpoint.