Imagexpress micro xl

Cells were fixed in 4 % paraformaldehyde (PFA) for 15 min and washed in PBS with 0.1 % triton X-100 (PBS-T). Cells were incubated with primary antibodies (Additional file 1: Table S1) overnight at 4 °C in immunobuffer containing 1 % goat serum, 0.2 % Triton X-100 and 0.04 % thimerosal in PBS. Cells were washed in PBS-T and incubated with appropriate anti-species fluorescently conjugated secondary antibodies overnight at 4 °C. Cells were washed again and incubated with Hoechst 33258 (Sigma-Aldrich, MO, USA) for 20 min. Images were acquired at ×10 magnification using the automated fluorescence microscope ImageXpress® Micro XLS (version 5.3.0.1, Molecular Devices, CA, USA). Quantitative analysis of intensity measures and positively stained cells was performed using the Cell Scoring and Show Region Statistics analysis modules on MetaXpress® software (version 5.3.0.1, Molecular Devices, CA, USA).

Pericytes were either pre-treated with MG132 (5 µM) for 6 h prior to incubation with α-syn ribbons or fibrils for 24 h. MitoSOX reagent was added to the cells at a final concentration of 5 µM. The cells were subsequently incubated for 30 min at 37 °C. 5% CO₂. To label cell nuclei, the NucBlue Live Cell counterstain was added at the same time as the CellROX reagent. The cells were subsequently imaged using the automated fluorescence microscope ImageXpress Micro XLS (Version 5.3.0.1, Molecular Devices, CA, USA) using the 20 x (0.45 NA) CFI Super Plan Fluor ELWD ADM objective lens and Lumencor Spectra X configurable light engine source. Quantitative analysis of several measures including percentage positive cytoplasmic staining, cytoplasmic intensity measures and granularity measurements were performed using the cell scoring, granularity and integrated morphometry analysis modules on MetaXpress software (Version 5.3.0.1, Molecular Devices, CA, USA). Roughly 500–1000 cells were scored per well with multiple wells (at least three) analysed per sample.

Microneutralization assays of SARS-CoV-2 virus were performed as previously described^{3 (link)}. The day before infection, Vero E6 cells were seeded at 1 × 10⁴ cells per well into 96-well plates. The diluted plasma and antibodies were mixed with SARS-CoV-2 WA1/2020 or the B.1.351 variant and incubated for 1 h at 37 °C. The antibody–virus mix was then directly applied to Vero E6 cells and incubated for 22 h at 37 °C. Cells were subsequently fixed by adding an equal volume of 70% formaldehyde to the wells, followed by permeabilization with 1% Triton X-100 for 10 min. After washing, cells were incubated for 1 h at 37 °C with blocking solution of 5% goat serum in PBS (catalogue no. 005–000-121; Jackson ImmunoResearch). A rabbit polyclonal anti-SARS-CoV-2 nucleocapsid antibody (catalogue no. GTX135357; GeneTex) was added to the cells at 1:1,000 dilution in blocking solution and incubated at 4 °C overnight. Goat anti-rabbit AlexaFluor 594 (catalogue no. A-11012; Life Technologies) was used as a secondary antibody at a dilution of 1:2,000. Nuclei were stained with Hoechst 33342 (catalogue no. 62249; Thermo Fisher Scientific) at 1 μg ml⁻¹. Images were acquired with a fluorescence microscope and analysed using ImageXpress Micro XLS (Molecular Devices). All experiments were performed in a biosafety level 3 laboratory.

Dil fluorescent dye-labeled acetylated low-density lipoprotein (Dil-ac-LDL; 10 μg/mL, Cell Applications) was added to phase IV medium of iECs at day 11 or day 21 and incubated for 4 h at 37°C, as described previously (24 (link)). Cells were then washed with PBS, fixed, and stained with DAPI. Images were taken with ImageXpress Micro XLS (Molecular Devices) and analyzed using ImageJ software. HUVECs fed with phase IV medium were used as positive control, and iPSCs fed with mTeSR⁺ medium were used as negative control.