Imagexpress micro xl automated

In total, 5 x 10³ U2OS cells were seeded into black 96-well µclear imaging plates (Greiner Bio-One) and allowed to adapt for 24 h. Thereafter, the cells were treated with DTT compounds and respective controls and incubated for additional 6 or 24 h before fixation in 3.7% (w/v) paraformaldehyde (PFA) in PBS supplemented with 1 µM Hoechst 33342 for 20 min. Upon fixation, cells were permeabilized with 0.1% Triton in PBS for 10 min at RT. Unspecific binding was blocked with 2% BSA in PBS for 10 min at RT followed by primary antibody diluted in BSA 2% following the manufactures recommendations overnight on an orbital shaker at 4 °C. The cells were rinsed twice and stained with AlexaFluor-coupled secondary antibodies for 1 h at RT, rinsed twice and subjected to imaging using an ImageXpress micro XL automated bioimager (Molecular Devices) equipped with a PlanApo 20 × /0.75 NA objective (Nikon).

U2OS cells or H4 cells stably expressing GFP‐LC3, RFP‐Lamp1, GFP‐TFEB, or GFP‐RFP‐LC3, or PC12 expressing Q74‐GFP were seeded in 384‐well black microplates and incubated for 24 h. After treatment, cells were fixed with 4% paraformaldehyde (PFA, w/v in PBS) for 20 min at room temperature and stained with 10 μg/ml Hoechst 33342 in PBS. Image acquisition was performed using an ImageXpress Micro XL automated microscope (Molecular Devices, Sunnyvale, CA, US) equipped with a 20 X PlanApo objective (Nikon, Tokyo, Japan). At least four view fields were acquired per well, and experiments involved at least triplicate assessment. Quantitation was usually done on 1,200–2,400 cells per condition. Upon acquisition, images were analyzed using the Custom Module Editor functionality of the MetaXpress software (Molecular Devices). Briefly, cells were segmented and divided into nuclear and cytoplasmic regions based on the nuclear Hoechst staining and cytoplasmic GFP or RFP signal. GFP‐LC3 dots were detected using automated thresholding, and their number and surface were measured in the cytoplasmic or/and nuclear compartment. GFP‐TFEB intensities were also systematically measured in both compartments. Data processing and statistical analyses were performed using the R software (http://www.r-project.org/).