Vybrant dii cell labeling solution

Late stage 46 Flk1:GFP transgenic tadpoles were injected in the midbrain ventricle with Vybrant DiI Cell-Labeling solution (ThermoFisher Scientific, #V2285). The tadpoles were immediately fixed in 4% paraformaldehyde in PBS, and left overnight at 4°C. Brains were dissected and whole mounted on coverslips (in 6 M urea in 50% glycerol) for confocal microscopy.

An amount equal to 2 μL of a 50 μM Vybrant™ DiI Cell-Labeling Solution (Thermo Fisher Scientific, Waltham, MA, USA) diluted in DMSO (Biolot, Saint-Petersburg, Russia) was added to 2 mL of the PPP followed by incubation at 37 °C for 20 min with moderate stirring. PURE-EV size exclusion chromatography columns (HansaBioMed, Tallinn, Estonia) were used for SEV purification/isolation according to the producer’s protocol, with slight modifications [23 (link)]. Briefly, 2 mL of the sample was loaded into the SEC column, and 23 fractions of 500 µL were collected. Fractions 9–11 were used for further experiments.

Transparent zebrafish eggs were dechorionated using 200 μg/mL pronase. After 24 hours of development, zebrafish embryos were arranged in agar injection plates and anesthetized with 2× tricaine, prepared from a 4 mg/mL stock and diluted in fish water. Cells were transiently labeled with Vybrant DiI Cell-Labeling Solution (ThermoFisher, V22885, Waltham, MA, USA) or Molecular Probes CellTracker^TM Green CMFDA (5-chloromethylfluorescein diacetate) Dye (Life Technologies, C2925, Carlsbad, CA, USA) to distinguish the different cell types (parental vs. reprogrammed vs. CRISPR-Cas9 CD133 knockdown). A 1:1 ratio of 100–200 fluorescently cells labeled with either Cell Tracker Green CMFDA Dye or vibrant DiI red cell-labeling solution, were injected into the yolk sac of the developing embryo using a glass microinjection needle. Around 100 to 200 fluorescent cells were injected into the embryonic yolk sacks of each of 30 anesthetized zebrafish embryos. After injection and recovery, each fish was transferred to a well in a 96-well plate. Fluorescent images of zebrafish were taken to assess overall fish health, injection success, and initial location of cells. Zebrafish were then imaged again under a fluorescence microscope at indicated times to assess cell metastasis characterized by cell migration to the tail.

For single-cell sequencing, EBs, Matrigel-embedded Epi rosettes and XEn/Epi EpiCs were manually picked and incubated in AccuMax solution at 37°C for 30 min and resuspended every 5 to 10 min to aid single-cell dissociation. For Matrigel-embedded Epi rosettes, the basal lamina gel was first removed by one time washing with PBS followed by Cultrex organoid harvesting solution (R&D Systems, 3700-100-01) or a 1:1 mix of DispaseII:N2B27 medium and incubated for 25 min at room temperature or 37°C, respectively. Dissociated cells were stained for a live staining (Vybrant DiI Cell-Labeling Solution, ThermoFisher, V22885) and dead staining (LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, ThermoFisher, L34975) to improve selection of viable cells. Gata6-h2b-Venus⁺ cells, which are indicative of PrE/VE, and non-fluorescent single cells were sorted in 384-well-plates for further RNA sequencing.

Murine RAW264.7 cells (ATCC; Manassas, VA, USA) were preloaded with Vybrant™ Di-I Cell-Labeling Solution (Thermo Fisher Scientific) according to the manufacturer’s instructions. Approximately 5 × 10⁴ cells/insert were placed in FluoroBlok™ 24-well cell culture light-blocking inserts with 8 µm pores (Corning, Corning, NY, USA) and allowed to equilibrate in normal culture media and conditions described above for 6 h. SCAP were cultured as described above at the concentration of 1 × 10⁵ cells/well in black-walled 24-well plates (Corning) in the presence or absence of 1 × 10¹⁷ heat-killed bacteria. After 2 days of culture, inserts containing the labeled macrophage cell line (RAW264.7) were transferred to the SCAP plates. After 24 h of co-culture, the fluorescence of the lower chamber was measured at 480 nm using a FlexStation 3 Benchtop Multi-Mode microplate reader (Molecular Devices, San Jose, CA, USA); then, representative images of fluorescently labeled cells were acquired using an EVOFL inverted microscope (Life Technologies) at 10× magnification.

Concentrated lipid stocks were prepared as previously described^{15 (link)}. Briefly, pure lipids were diluted in chloroform and dried in acid-washed glass centrifuge tubes under a stream of nitrogen. Phospholipid samples were suspended at 2–6 mM in phosphate-buffered saline at pH 7.2 and sonicated twice for 5 min at power setting 0.2–0.5% amplitude. All samples were sterilized with 0.22 µm-pore filters (Sartorius). The recovery of phospholipids after filtration was typically 90% or more. The Dynamic light scattering (DLS) analysis revealed and overage diameter of 127 ± 18 nm for Ptdcho and 82 ± 27 nm for PtdEtn liposomes (Supplementary Fig. 5). In addition, we have evaluated by thin layer chromatography (TLC) that the major lipid present in the filtrated solution are liposome-containing phospholipids, and thus, discarding the presence of phospholipids-hydrolyzed species like lysophospholipids (Supplementary Fig. 5). Diluted phospholipids were added to the growth medium at different concentrations, as described throughout the text. The fate of liposome was evaluated by measuring the incorporation of red fluorescence in cell treated with liposome-labeling with Vybrant™ DiI Cell-Labeling Solution (Thermo Fisher) (Supplementary Fig. 5).