μ slide chamber

For activation experiments on glass without SLB, 8-well μ-slide chambers (Ibidi) were coated with either PLL or anti-CD3/CD28 prior to cell loading. PLL was applied by incubation of 250 μl/well 0.01% PLL (Sigma Aldrich) in dH₂O for 15 min followed by 3 washes with 300 μl PBS. For antibody coating, wells were first coated with 250 μl 50 μg/ml polyclonal donkey anti-mouse antibody (ThermoFisher Scientific) in coating buffer (50 mM Na2CO3, 50 mM NaHCO3, pH 9.6, filtered using a 0.22 μm Millex®-GP syringe filter unit) at 4°C overnight, then washed with 3 x 300 μl PBS and incubated with 250 μl mouse anti-CD3 (OKT3; BioLegend) and mouse anti-CD28 (CD28.2; eBioscience) at 5 μg/ml in PBS for 1 h before final 3 × 300 μl PBS washes.

U87EGFRvIII cells labeled with the long term Qtracker705 dye (Molecular Probes), were co-cultured with U87EGFRwt cells in a ratio of 1:9. After 2 weeks of co-culture, neurospheres containing both phenotypes were formed. Nuclei were stained with Hoechst 33342. The neurospheres were seeded in μ-Slide chamber (Ibidi) pre-filled with ice cold liquid Cultrex (basement membrane matrix, mentioned as matrigel in the main text, 40% in DMEM, Trevigen). After 30 min at 37 °C the matrigel has gelified, trapping the neurospheres. Each neurosphere was imaged using a 10×, 0.3 dry EC Plan-Neofluar objective mounted on a ZEISS Confocal LSM710 microscopy. Z-stacks were obtained using 3 μm step size along the axial axis with 80–130 slices in total. During imaging (24 h), cells were maintained at 37 °C and 5% CO2. XYZ coordinates for each cell nucleus were determined using IMARIS 7.2.3. (BITPLANE), and cell–cell separation distances distributions were calculated as described above. All pairs having a distance up to 800 μm were processed as described above.

Monocytes were isolated from the peripheral blood mononuclear cells layer obtained after histopaque centrifugation and purified using a percoll solution as described previously^{27 (link)}. The chemotaxis of monocytes against the EVs was determined using a μ-slide chamber (ibidi) following the manufacturer’s instructions. One side of the chemotaxis chamber was loaded with the EVs, and monocyte were allowed to migrate. For comparison, one side of the chemotaxis chamber was loaded with MCP-1, and monocyte chemotaxis was assessed. For the inhibition of monocyte chemotaxis against EVs, monocytes were pretreated with CCR2 inhibitor (1 μg/ml, Santacruz) for 30 min and allowed to migrate toward EVs in the presence of the CCR2 inhibitor. The cell movements were visualized and captured by the Nikon Eclipse Ni-U microscope using × 20 objective. The chemotaxis was then analyzed using ImageJ and chemotaxis/migration tool (ibidi).

Neutrophils (5 × 10⁶ cells) were stained cell tracker green (1.5-2.0 μg/mL, Invitrogen) and incubated in either μ-slide chamber (Ibidi) pre-coated with fibronectin (5 μg/mL, Merck Millipore) for evaluation of NDTR formation or in confocal plates for evaluation of NDMV formation. Neutrophils were stimulated with various stimulators: fMLP (1 μM, Sigma-Aldrich), LPS (1 μg/mL, Sigma-Aldrich), C5a (50 ng/mL, Sino Biologicals), S100B (100 ng/mL, Sino Biologicals), HMGB1 (100 ng/mL, Sino Biologicals), TNF-α (50 ng/mL, Sino Biologicals), IFN-γ (100 ng/mL, Sino Biologicals), TGF-β (20 ng/mL, Sino Biologicals), IL-4 (20 ng/mL, Sino Biologicals), PMA (100 μg/mL, Sigma-Aldrich), and N omega-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 10 μM, Tocris bioscience). Then, EV formations were visualized by immunofluorescence microscopy (Olympus IX83, Olympus) at 37 °C, 5% CO₂ for 1 hour.

Migration of monocytes against neutrophil-derived EVs were determined using μ-slide chamber (ibidi) following manufacturer's instruction. One side of chemotaxis chamber was loaded with either NDTRs or NDMVs, and monocytes were allowed to migrate. For comparison, chemotaxis chamber was loaded with MCP-1 (100 μg/mL), and migration of monocytes were measured. For inhibition of monocyte chemotaxis against EVs, cells were pre-treated with CCR2 (a receptor for MCP-1) inhibitor (CCR2 inhibitor, 1 μg/mL, Santa Cruz) for 30 min and allowed to migrate toward EVs in presence of CCR2 inhibitor. The cell movement were visualized and captured by Nikon Eclipse Ni-U microscope using 20X objective. The movement of cells was then analyzed using ImageJ 68 (link) and Chemotaxis/migration tool (ibidi).