Planapo 20x 0.75 na objective

Five x 10³ U2OS cells stably 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 Lytix-315 and respective controls and incubated for additional 6 or 24 h before fixation in 3.7 % (w/v) paraformaldehyde 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 2 % BSA in PBS shaking over night 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 20X/0.75 NA objective (Nikon). For some of the assays cells were additionally stained with 200 nM MitoTracker green or MitoTracker orange (Life Technologies), 1 ug/mL FITC-coupled Phalloidin before imaging.

Five x 10³ U2OS cells stably expressing PARKIN-RFP; SMAC-GFP; LC3-RFP; TFAM-GFP; BAX-GFP or Mito-RFP 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 LTX-315 and respective controls and incubated for additional 6 or 24 h before fixation in 3.7 % (w/v) paraformaldehyde in PBS supplemented with 1 μM Hoechst 33342 for 20 min. Upon fixation, PFA was substituted with PBS and a minimum of four view fields per well were acquired by means of an ImageXpress micro XL automated bioimager (Molecular Devices) equipped with a PlanApo 20X/0.75 NA objective (Nikon). For some of the assays cells were stained with 200 nM MitoTracker green or MitoTracker orange (Life Technologies), 1 μg/mL FITC-coupled Phalloidin before imaging.

Fluorescence microscopy was performed as described (59 (link)). Mycobacterial cording and macrophage numbers were assessed in the trunk of the larvae using a Nikon Eclipse E600 upright microscope fitted with Nikon Plan Fluor 10X 0.3 NA and Nikon Plan Fluor 20X 0.5 NA objectives. For laser scanning confocal microscopy, anesthetized larvae were embedded in low-melting-point agarose as previously described (6 (link)). A Nikon A1R confocal microscope with a Plan Apo 20X 0.75 NA objective was used to generate 35-40 mm z-stacks consisting of 0.3-2-mm optical sections. The galvano scanner was used for all static imaging and for time-lapse imaging of the caudal hematopoietic tissue (CHT, area located between the cloaca and the beginning of the caudal fin). Images were acquired with NIS Elements (Nikon). A heating chamber (Oko-labs) adapted to the microscope was used to maintain temperature at 28.5°C during imaging. Confocal images are pseudocolored to facilitate visualization.