Oregon green 488

The dimeric N constructs with removed cellular nucleic acids were labeled by fluorescence dye Oregon-Green488 (Invitrogen, 2,161,802) with a molar ratio 10:1 between protein and fluorescence dye in labeling buffer 50 mM NaPhosphate pH 7.0, 50 mM NaCl. The mixtures were incubated at 25 ℃ for 1 h. To remove the excess fluorescence dye, the resulting mixture was buffer-exchanged with labeling buffer using concentrator columns with 10 kDa cutoff (Ultrafiltration Centrifugal Tube, Millipore). The SARS-CoV-2 5' UTR and 3' UTR were labeled by fluorescence dye Cy3 (Lumiprobe, #41,070) following the Cy3 labelling protocol [79 (link)]. The labeled proteins and RNAs were quantified using a NanoDrop One^C (Thermo Scientific).
Oregon-Green488 labeled N constructs were diluted to a final concentration of 25 μM in phase separation buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl) containing 10% PEG 3350, and RNAs were added with a protein to RNA molar ratio of 100:1. Then the mixture was incubated at room temperature for 5 min. A total of 10 μL solution was transferred onto the glass slide, and images were collected using a Zeiss-Axio Observer 7 microscope.

C-SNARF-4 calibration samples were prepared by mixing 10 μl of 1 mM C-SNARF-4 in DMSO stock solution with 90 μl of 100 mM sodium citrate buffer adjusted to pH 3.0–7.0. A 20 μl drop of the solution was placed on a glass coverslip and imaged with an inverted confocal microscope.
C-SNARF-4 stained cheese samples were prepared by cutting a small piece (about 5 × 5× 0.5 mm) from the bulk of the cheese, the piece was then placed on a glass coverslip with a 20 μl drop of fluorophore solution (10 μl of 1 mM C-SNARF-4 in DMSO stock solution mixed with 90 μl of deionized water). Additionally, cheese samples without the fluorescent probe were prepared to assess the amount of cheese autofluorescence.
Oregon Green 488 (Molecular Probes, D-7172) stained calibration samples were prepared by immersing a small piece of cheese in a 40 μl drop of Oregon Green 488 buffered solution (20 μl of 50 μM dye in deionized water, mixed with 20 μl of 100 mM sodium citrate buffer adjusted to pH 3.0–6.0) on a glass coverslip and imaged with an inverted microscope.
For cheese pH measurements the same protocol was followed, but the citrate buffer was replaced by an equal amount of deionized water to maintain the same dye concentration as in the calibration samples. To assess the cheese autofluorescence, cheese samples without the fluorescent probe were also prepared.

EVs from MCF7 and MDAMB 453 were prepared from Wheat-germ-agglutinin (OregonGreen 488, Invitrogen, USA). They were seeded at a high concentration in super-resolution mounting medium (ProLongGlass, Invitrogen) over 1.5 thickness super-resolution cover-glasses for acquisition in both laser scanning confocal microscopy (LCSM) using a Nikon EZ-C1 instrument with multi-spectral head and argon Laser, equipped with a 1.40 NA 100x oil objective, and in super-resolution structure illumination microscopy (SIM) using the 2D SIM/TIRF modality with total internal reflection angle illumination using high power 488 laser and 2D-grid SIM detection followed by FFT Gustaffson algorithm reconstruction over a Nikon N-SIM/N-STORM instrument, equipped with 1.49 NA 100× TIRF oil objective. Reconstructed images were analyzed using NIS-Elements V.5.31 software (Lim/Nikon Instruments) for image quantification, using an ad hoc implemented pipeline of image processing and segmentation (GA3 module in NIS-Elements). Data were further elaborated with GraphPad PRISM v.9.