Ibitreat slide

Experiments to investigate the intracellular localization of NPs were performed in Lab 2 (Mainz) and Lab 3 (Jena) using confocal laser scanning microscopy (CLSM). For these experiments the HepG2 cells were seeded in ibiTreat µ-slides (IBIDI, Germany) (Lab 2, Mainz) or in glass chamber slides (BD, Germany) (Lab 3, Jena). After 24 h, NPs were added to the medium at a concentration up to 500 µg/mL. Before CLSM imaging cells were washed with PBS. Live cell images were taken in Lab 2 (Mainz) with a commercial setup (LSM SP5 STED Leica Laser Scanning Confocal Microscope, Leica). There, the cell membranes were stained with CellMaskOrange (2.5 mg·mL⁻¹, Invitrogen), and the cell nucleus with DraQ5 (2.5·10⁻⁶ M, Biostatus). In Lab 3 (Jena), for microscopy observations with the LSM 510 Meta (Carl Zeiss MicroImaging GmbH) cell membrane was stained with Wheat Germ Agglutinin Alexa Fluor® 633 conjugate (Invitrogen) and the nucleus with Hoechst 33258 (Applichem) after fixation of cells with 4% formaldehyde. Fluorescent particles were detected at 533–570 nm. For a control experiment to investigate the clustering and adhesion of the NPs to differently coated culture slides, the SiO₂ NPs were suspended in complete culture medium by pipetting and then they were added to the slides without any cells for supplementary microscopy studies.

Blastocysts at E3.5 were cultured for 24 hr at 37 °C and 6% CO₂, in ibiTreat µ-Slides (Ibidi GmbH, Munich, Germany) on feeder cells that were previously growth-arrested with a 2 hr mitomycin C treatment (10 μg/ml, Sigma-Aldrich), in mESCs derivation media mESCs media except with the FBS replaced with Knockout Serum Replacement (15%, Thermo Fisher Scientific). The cultures were fixed and processed for IF staining as described above for the E3.5 blastocysts. Slides were mounted with VectaShield mounting medium (Linaris; Dossenheim, Germany).

Gliding properties of the wild type and mutant strains were analyzed by time-lapse microcinematography as previously described^{5 (link)}. Samples from mid-log phase cell cultures were grown overnight on eight-well ibiTreat µ-slides (Ibidi, Gräfelfing, Germany) filled with 200 µl of SP4 medium. Prior to the observation, medium was replaced with fresh SP4 pre-warmed at 37 °C and cell movement was examined at 37 °C and 5% CO₂ using a Nikon Eclipse TE 2000-E inverted microscope equipped with a CO₂ Microscope Cage Incubation System (Okolab, San Bruno, USA). Images were captured at 2 s intervals for a total of 2 min for all different strains. The frequency of motile cells was determined by examining approximately 250 isolated cells of each strain. Given that cells of the P110-W838F mutant were mainly aggregated, only 64 individual cells could be analyzed. Mean velocities were obtained from the analysis of at least 25 motile, isolated cells, by measuring the traveled distance and dividing this value by the time spent in microcinematography. Analysis of the gliding properties was performed using the ImageJ software with the MTrack2, MTrackJ, wrMTrack, and Stack Deflicker plugins (https://imagej.nih.gov/ij/).

HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS and 1% PenStrep. They were seeded into ibiTreat µ-slides (ibidi) at a concentration of 5000 cells per well in 300 µL of DMEM. The day after seeding, cells were incubated with particles for 24 h and for the lysosome experiments also with 8 µL of CellLight Lysosomes-GFP (Thermo fisher scientific, Waltham, MA, USA). The particles were labeled with Atto633-NHS overnight and then prepared as for the characterization described above. For internalization experiments cells were stained with WGA488 (Thermo fisher scientific, Waltham, MA, USA), and washed with DMEM prior to imaging. For imaging we used a spinning disk microscope (Zeiss Cell Observer SD with a Yokogawa spinning disk unit CSU-X1). Lysosome-GFP and WGA were excited with a 488 nm laser and the particles with a 639 nm laser. Band-pass filters 525/50 and 690/60 (both Semrock) were used in the detection path for Lysosome-GFP/WGA and the particles, respectively.

MΦ migration was assessed using a scratch assay and live cell tracking of MΦ on fibronectin. For the scratch assay, 1.5 × 10⁵ MΦ in 6-well plates were transfected with mimic control (MISSION^® miR negative control 2 (cel-miR-39a; Sigma-Aldrich)) or hsa-miR-200c-3p mimic (MISSION^® hsa-miR-200c-3p mimic). After 24 h, a 10 µL pipette tip was used to scratch the MΦ layer in a marked area. Pictures were taken every 24 h using a Canon EOS 600D camera (Canon, Ōta, Tokio, Japan) and a transmitted-light microscope (AxioVert 40; Zeiss, Oberkochen, Germany). The scratch area was evaluated using ImageJ software. To observe random MΦ migration on fibronectin, 2 × 10⁴ miR-200c mimic-transfected MΦ were transferred onto fibronectin (1.5 μg/cm², fibronectin human plasma, Sigma Aldrich)-coated 8 well ibiTreat µ-slides (ibidi GmbH, Gräfelfing, Germany) 48 h after transfection. The coating was achieved following the manufacturer’s instructions. Live cell tracking was performed on a Cell Observer microscope (Zeiss) at 37 °C and 5% CO₂ for 16 h with pictures taken every 10 min. Analysis of cell migration was performed using the tracking application of the AxioVisionSoftware with 50 cells tracked per condition of each replicate. Migration plots were generated for 15 representative tracks using the Chemotaxis and Migration Tool from ibidi.