In cell developer toolbox 1

Quantification of dopaminergic and cholinergic phenotypes was performed using high-content analysis in control cells and cells exposed to SiPCL-NPs on DIV1, DIV4 and DIV6. The SH-SY5Y cells were seeded in a 96-well microplate at a cell density of 8.4 × 10³ cells per well. On DIV7, the cells were fixed and blocked as described above. The primary antibodies against TH and CHT1 (see Table 1) were diluted in a stock solution consisting of 0.4% Triton-X PBS with 2.5% horse serum, and the incubations with the cells were performed overnight at 4 °C.
As described above, the secondary antibodies (Table 2) were applied together with Hoechst 33342 nucleic acid stain on the following day. For the quantitative analysis of TH- ir and CHT1-ir cells, high content analysis of the SH-SY5Y cells was performed with an INCell Analyzer 2000 (INCA) (General Electric Healthcare Europe GmbH, Glattbrugg, Switzerland).
Images were taken from three wells per group and 35 randomly assigned areas for each well. The total number of cells, evaluated by the number of nuclei, and TH- or CHT1-ir cells were determined using the INCell Developer Toolbox 1.9.2 (General Electric Healthcare Europe GmbH, Glattbrugg, Switzerland).

CHSE-214 cells were fixed with PFA diluted at 4% in PBS followed by a second fixation with cold methanol. Each fixation step lasted 15 minutes. Cells were washed with PBS after each fixation step. Blocking buffer containing 5% goat serum (Sigma-Aldrich) and 0.3% Triton X-100 (Sigma-Aldrich) was added to each well with the cells for 1 hour. Then, anti-VP3 2F12 antibody was diluted 1/500 in antibody dilution buffer (1% BSA (Sigma-Aldrich), 0.3% Triton X-100) and was incubated for 1 hour. FITC-labelled goat anti-rabbit was used as secondary antibody at 1/300 dilution. Cells were washed three times after each antibody incubation with PBS. IF images were taken INCell Analyzer 6000 imaging system.
IN Cell Developer Toolbox 1.9.2 (RRID: SCR_015790; GE Healthcare, Little Chalfont, UK) was used to count number of IPNV foci (positive areas after image segmentation were selected when >21000 fluorescence units and >2500 µm
² criteria was reached).

Following the staining procedure, plates were imaged with a high-content imaging system (Operetta, PerkinElmer, Rodgau, Germany) at 10× magnification, nine fields per well. Images were analyzed to extract the total tube length and branching point parameters using Metamorph (Molecular Devices, San Jose, CA, USA) image analysis software. We used an automated sequence in the IN Cell Developer Toolbox 1.9.2 software (GE) to quantify the lumen area of the vascular-like structures (Figure S2). To observe the dynamics of vascular-like structure formation, the time-lapse live imaging was performed using GFP lentiviral-transduced HUVEC in a heated and CO₂-controlled and integrated chamber within the high-content imaging system as described previously [64 (link)]. Images were captured with 10× objective at 15 min intervals for a total of 16 h.