In vivo intravital microscopy was performed using the Nikon TiE inverted LSM C2 confocal system. A custom-heated stage was designed for intravital imaging (Okolab, NA, Italy). To limit movement during intravital imaging, the machine shop at UT MD Anderson Cancer Center Radiation Physics custom-built a window chamber holder. The stage temperature was set to 37°C to continuously warm animals during image acquisition. Mice were kept under isoflurane anesthesia using an isoflurane vaporizer with an active evacuation (XGI-8 PerkinElmer/Caliper Life Sciences, Waltham, MA). To further minimize stray light during acquisitions, a small black box with a notch cut out for the anesthetic nose cone was placed over the animals. For in vivo imaging of MDCs, dextran, Texas Red (10,000 molecular weight, 4 mg/ml; Thermo Fisher Scientific, Waltham, MA), and Gr-1 antibody conjugated to Alexa Fluor 647 (10 μg; BioLegend, San Diego, CA) were intravenously administered to label MDC through a catheter in the tail vein. Tumor cell dendra2 as well as MDC Texas Red and Alexa Fluor 647 were imaged using the Nikon TiE inverted LSM C2 confocal system with the following image acquisition parameters: 4.8 speed on a 2× or 10× objective using laser lines of 488 nm (dendra2 excitation), 535 nm (Texas Red excitation), and 640 nm (Alexa Fluor 647 excitation). Live sequential C2 confocal imaging was performed before intravenous injection of dextran and Gr-1 antibody injection, and image acquisition continued during administration of dextran and Gr-1 antibody to confirm successful intravenous injection. The total sequential C2 confocal acquisition time was 10 min. An ROI was determined from the 2× confocal images for imaging with the 10× objective. After successful intravenous injection of dextran and Gr-1 antibody, an intraperitoneal injection of d-luciferin (300 mg/kg) was administered to animals, and 5 min after administration, a BLI was acquired using the following parameters: 10× objective, sensor gain set to 4×, read speed of 50 kHz, and binning 2 using a 20-min exposure. A C2 confocal image of the same ROI using a 10× was acquired with the same acquisition parameters described above and a Z stack acquisition of 110 slices at 1-μm steps. After intravital imaging, animals were given a bolus injection of 1 ml of saline and allowed to recover in their cage on a heating pad.

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