Celltrackertm cm dii

Fish of the strain Tg(fli1a:EGFP)y1 were raised and maintained according to the National (Italian D.lgs 26/2014) and European (2010/63/EU and 2012/707/EU) animal guidelines. Embryos were collected by natural spawning and staged according to Kimmel and colleagues (Kimmel et al, 1995 (link)). GH3 cells, infected with unspecific shRNA (shCtrl) or with shAngpt2 were labeled with a viable red fluorescent dye (CellTrack‐erTM CM‐DiI, Invitrogen), resuspended in PBS and implanted into the sub‐peridermal space of embryos at 48 h postfertilization. At 24 and 48 h post‐injection, embryos were imaged through a fluorescence stereomicroscope (Leica DM6000B equipped with LAS Leica imaging software). In some pictures, the red channel was omitted to highlight the differences of tumor‐induced microvascular networks between the two experimental groups. Digital magnifications of graft regions are provided and all images shown are oriented so that rostral is to the left and dorsal is at the top. The same exposure time was used for all panels. At least 20 correctly grafted embryos have been included in each experimental group. As arbitrary unit (A.U.) of the tumor‐induced angiogenesis, the total cumulative length of endothelial structures sprouting from the SIV and CCV was measured in each grafted embryo by Fiji software and expressed in number of pixels.

hCSCs were labeled for tracking with CellTracker^TM CM-DiI (Invitrogen #C7000). For the labeling process, we suspended the cells at 1 × 10⁶ cells/mL in DPBS and added DiI at 1 µM/mL. After incubation for 5 min at 37 °C and then 15 min at 37 °C, the cells were centrifuged at 1500 rpm for 5 min and washed twice with DPBS. To prepare cells for transplantation, DiI-labeled hCSCs (1 × 10⁶) were suspended in 100 μL of saline.

For the evaluation of EV biodistribution at 2 hours after intravenous administration, an additional six animals were used, which were divided into two groups: sham group (n = 3) and TMEV-EVs (n = 3). TAPA1 marker levels (CD81) (1:750, Abcam) were observed in the brain as well as in samples from the peripheral organs, such as the lungs, spleen and liver. A colabelling of EVs (CD63) with glial fibrillary acid protein (GFAP) (1:400, Millipore) and neuronal specific nuclear protein (NeuN) (1:100, Millipore) was made by immunofluorescence. The EV phenotype was determined by immunofluorescence with a specific lipophilic tracer, CellTrackerTM CM-DiI (Invitrogen, Carlsbad, CA, USA), and then colabelled with ALIX (1:250, Abcam). Images were acquired as a maximum confocal projection using a LEICA TCS SPE spectral confocal microscope (Leica Microsystems, Heidelberg, Germany) and analysed using LAS AF software (Leica). Mean fluorescence intensity was measured by the NIS-Element AR (Nikon) 4.5 Program.

On day 30 of lung organoid differentiation, hPSC-derived macrophages were stained with CellTracker^TM CM-DiI (C7000, Invitrogen, Waltham, MA, USA). Live staining was performed according to the manufacturer’s instructions. The macrophages were then injected into the lung organoid at a concentration of 2.0 × 10⁷ cells/mL using a 0.3 mL insulin syringe. Fluorescence was continuously observed after the injection on day 45 for organoid differentiation. During the macrophage injection stage, the cell-cultured medium used a “lung organoid medium”.

2 × 10⁵ human umbilical vein endothelial cells (HUVECs) were seeded in each well of a four-well plate 48 h before the assay to prepare a monolayer of ECs. Then, 1 × 10⁵ EPCs labelled with CM-DiI (CellTrackerTM CM-DiI, Invitrogen) were added to each well and incubated for 3 h at 37°C. Nonattached cells were gently washed and removed with PBS, and adherent EPCs were fixed with 4% paraformaldehyde and counted by independent investigators blinded to treatment randomly.