Imaris software v 9.0.1 64

All experiments were performed at least three times. Represent images were placed in figures. Primary image data which was analyzed and calculated by Leica LAS AF software (Leica Microsystem, Inc) or Imaris software v.9.0.1 64× (Oxford Instruments plc) was acquired and processed with Microsoft Excel software. Error bars with ± SEM, and p values were calculated with unpaired t-test in GraphPad Prism 9.01 (GraphPad software). p<0.05 was considered significantly different.

STED microscope imaging for VLPs using modified protocol from previous report (Chojnacki et al., 2012 (link)). Labeled recombinant MFG-E8-Alexa Fluor 594 (250 ng) was incubated with NL4.3-GFP VLPs (0.1 × 10⁶ of GFP positive particles) at 4°C for 30 min. Ten micro liters of MFG-E8/NL4.3-GFP mixture mixed with 40 μl of mounting solution (ProLong Gold, Life Technologies). Ten micro liters of final mixture was mounted on a slide covered with #1 coverslip. All STED imaging was performed with a Leica DMI6000 SP8X CW gated STED system with a 775 nm depletion laser (STED dichroic slider) and an HC PL APO CS2 100× (NA, 1.40) oil objective. Pixel size was set to 19 nm. Images were acquired with Leica LAS AF software (Leica Microsystem, Inc) and processed Imaris software v.9.0.1 64× (Oxford Instruments plc). Raw images were filtered with Gaussian filter which is standard function of Imaris software. The intensities of fluorescent signals in regions of interests (ROI) were measured by LSA AF Lite software (Leica Microsystem).

HEK293T cells were transfected using TransIT-293 Transfection Reagent (Mirus Bio LLC). Same transfection protocol with VLP preparation was applied for imaging experiments. Transfected HEK293T cells were imaged with a Leica SP8 inverted five channels confocal microscope (Leica Microsystems) equipped with 40 × oil objective, 0.95 NA (immersion medium used distilled water). The temperature of air (5% CO₂) was maintained at 37.0 ± 0.5°C. VLPs were detected by GFP signals. MFG-E8 was visualized by antibody (1 μg/ml) addition into culture media. Fluorescent MFG-E8 (250 ng/ml) which was added into culture media visualized by its own signal. For NL4.3-GFP/MFG-E8 capture experiment, eFluor450 (Thermo Fisher Scientific) labeled b12HL B cells were directly added into culture of HEK293T cells and imaged. Images were acquired with Leica LAS AF software (Leica Microsystem, Inc) and processed Imaris software v.9.0.1 64× (Oxford Instruments plc). Raw images were filtered with Gaussian filter which is standard function of Imaris software.