Nis elements c 4

THP-1 monocytes (1 × 10⁵ cells per well in 100 μl of cell culture media) were seeded on 96-well cell culture plates (Sarstedt) and incubated with 10 μg/mL EVs for 1 h at 37 °C. Following incubation, the cells were washed in 1 mL PBS and centrifuged at 2000 rpm for 5 min. The cells were then fixed with 4% paraformaldehyde solution at room temperature for 20 min and washed twice with 1 mL PBS. Fixed cells were stained for F-actin filaments with ActinRed 555 ReadyProbes reagent (Invitrogen, USA), and cell nuclei were counterstained with Hoechst 33,342 (Invitrogen, USA). Following staining, samples were washed three times with 1 mL PBS. Stained cells were smeared on adhesion microscope slides (Marienfeld, Germany), air-dried at room temperature for 10 min to remove excess water and then mounted with ProLong Diamond (Invitrogen, USA). Mounted cells were visualized under a Nikon C2 microscope using a FITC filter for Syto RNA Select, a TRITC filter for ActinRed 555 ReadyProbes reagent and a DAPI filter (Nikon, Japan) for Hoechst 33,342. Each channel was recorded separately to avoid spectral overlap. The images were analyzed using Nis-Elements C 4.13 software (Nikon, Japan). The EV uptake by fluorescence microscopy was studied in 3 biological replicates.

For the z-scan measurements, MSC spheroids were stained with 5 μM 3,3’-dioctadecyloxacarbocyanine perchlorate (DiO) in complete medium for 45 min at 37 °C and counterstained with Hoechst dye. The cells were then trypsinised and seeded in 3D culture for 24 h. The spheroids were then transferred to 4-well chamber slides. All reagents were obtained from Thermo Fisher Scientific, Waltham, Massachusetts, USA.
Fluorescence imaging was performed as described previously [54 (link)]. A Nikon eclipse Ti microscope equipped with a Nikon C2 confocal system was used. A Nikon S Plan Fluor ELWD 40×/0.60 objective was used. A 488 nm laser was used to excite CD90 FITC and DiO. In addition, a 405 nm laser was used to excite Hoechst and QD655. To detect the fluorescence, the following filters were used: 447/60 nm with Hoechst 525/50 nm for CD90 FITC and DiO, and a 561 long pass filter for QD655 (all from Nikon, Tokyo, Japan). Each channel was recorded separately to avoid spectral overlap. The images were analysed using Nis-Elements C 4.13 software (Nikon, Tokyo, Japan). Quantification of the QD fluorescence signal was performed using Nis-Elements C 4.13 software.