Plan fluor objective

A total of 10 µL of 0.1 mg/mL Poly-d-Lys solution was added to 384-well plates (Corning #3712) for 5 min, followed by a PBS wash. Plates were dried for at least 2 h; 50 µL of 600000 HEK293 GFP cells/mL of DMEM was dispensed into each well. After 24 h incubation to allow the cells to adhere, 200 nL of small molecules (or DMSO in columns 1, 2, 23 and 24) were pinned. Following a 16-h incubation, 200 nL of 5 mM CCCP was added to all wells except columns 1 and 2, into which 200 nL of DMSO was added. Following 2-h incubation, cells were fixed with 4% paraformaldehyde (PFA) and stained with 50 µL of 1 µg/mL DAPI solution for 15 min. After the final PBS wash, plates were ready for high content microscopy.
Images were acquired on IN Cell Analyzer 6000 (GE Healthcare), equipped with sCMOS camera (2048 × 2048), and 20×/0.45 NA Plan Fluor objective (Nikon) in open aperture mode using 1 × 1 binning. Image analysis was performed using Columbus Image Analysis System (PerkinElmer). Nuclei were initially detected in DAPI channel, followed by whole-cell segmentation in GFP channel. Using PhenoLOGIC machine learning plug-in, cells were categorized into two sub-populations: cells with even GFP-Parkin distribution and cells with GFP-Parkin localized to mitochondria. The percentage of cells with mitochondrial GFP-Parkin was determined per well and averaged across two independent trials.

Devices were illuminated with a Mercury-100W mercury lamp (Chu Technical Corporation) and imaged using a Nikon Eclipse TE300 inverted microscope, outfitted with 4× Plan Fluor, 10× and 20× Plan Fluor objective lenses (Nikon). Images were collected using a SPOT RT Monochrome camera (Spot Imaging Solutions, Diagnostic Instruments Inc.) and recorded with the manufacturer’s Spot 3.5 Advanced software.

Sample and guide fluids were loaded into 1-ml Primo syringes (Codan Medical ApS, Rødby, Denmark) and 5-ml Hamilton syringes (Hamilton Laboratory Products, Reno, USA), respectively, and delivered into the microfluidic device using neMESYS precision syringe pumps (CETONI, Korbussen, Germany). The microfluidic device was mounted on an Eclipse TS100 inverted microscope (Nikon, Zürich, Switzerland) equipped with a EOSens 3CL high-speed camera (Mikrotron, Unterschleissheim, Germany) for bright-field imaging and a CoolSNAP HQ2 charge-coupled device (CCD) camera (Roper Scientific, Ottobrunn, Germany) for fluorescence imaging. Bright-field images were acquired using a MCWHD3 white light LED (Thorlabs, Dachau, Germany) in combination with a 20×, 0.45 numerical aperture (NA) S Plan Fluor objective (Nikon, Zürich, Switzerland) and a 0.7× demagnification lens (yielding a total magnification of 14×). Fluorescence measurements were conducted using an Intensilight C-HGFI mercury light source (Nikon, Zürich, Switzerland) as an excitation source combined with tetramethylrhodamine filter set (AHF Analysentechnik, München, Germany), with emission being collected with a 10×, 0.3 NA Plan Fluor objective (Nikon, Zürich, Switzerland). Images were then processed using ImageJ (U.S. National Institutes of Health, Bethesda, USA).

Prior to differentiation, mitoLUHMES cells were mixed with wild-type LUHMES cells in proportion of 1:200. This allowed performing microscopy imaging of the single neurons in high-density culture. Cells growing in pre-coated 8 well Lab-Tek II chambers were treated with vehicle medium or medium containing 100 μM 6-OHDA or 10 μM CCCP. Living cells were imaged by fluorescence microscopy with the use of NIKON Eclipse Ti microscope equipped in ×60 oil-immersed Plan Fluor objective (Nikon), DS-Qi1 Nikon monochrome CCD camera (1.4 numerical aperture and 1.515 refractive index), and incubator with automatic monitoring of temperature and CO₂ levels (Oko-Lab). For confocal images, we used Nikon A1 confocal; and 488 argon laser was used to excite GFP emission. For 3D imaging, a water immersed ×60 NIKON objective was used.

For quantitative assessment of apoptotic cells, HEK293 and HeLa cells were seeded onto a 96-well plate and transiently transfected with the different forms of the short isoform of ABCG1. Twenty-four h later, the cells were incubated in Annexin V binding buffer containing 20-fold diluted Alexa Fluor 488-labeled Annexin V and 5 μM Hoechst33342 (Life Technologies/Thermo Fisher Scientific, Waltham, MA USA) for 5 min. Images were acquired by the ImageXpress Micro XLS wide-field high-content screening system (Molecular Devices) using a 10× Plan Fluor objective (Nikon, NA = 0.3). Twelve separate fields of view in each well were imaged for sampling the total cell culture. Using MetaXpress software, Annexin V-labeled cells and the total cell number were counted on the basis of green and blue fluorescence, respectively. Single cells were counted on the basis of Hoechst33342, a nuclear dye, with limits set at minimum and maximum width (10–25 µm) and minimum intensity difference from the background (1000 au). Next, Annexin V positive cells were identified, considering the nuclear staining to exclude cell debris without nuclear staining from the analysis. The results were expressed as the mean of a percentage of the total cell number ± S.E.M. For statistical analysis, a Student’s t-test was used to evaluate significant differences. (p < 0.05) in comparison with controls.