Ultraview vox spinning disk confocal system

Astrocytes were seeded in MatTek dishes 48 h before experiments. Cells were treated with 5 mM NH₄Cl or water for 72 h. After treatment, cells were stained with 1 μM LysoSensor™ Green DND-189 (Invitrogen) for 30 min. For NH4Cl-treated group, 5 mM NH4Cl was present during the staining procedure. Astrocytes were then imaged with the same setting using the spinning disk confocal microscopy (Eclipse Ti microscope (Nikon) and UltraVIEW vox spinning disk confocal system (PerkinElmer)).

Cells were plated on glass-bottom dishes (P06G-1.5-20-F, MatTek) in full medium overnight and were then imaged by time-lapse microscopy at 37 °C and 5% CO₂ in a live-cell incubation chamber. LLOMe (300 nM) was added to the medium, and cells were imaged for 1 h at 15-min time intervals, as indicated. For LysoTracker staining experiments, MEFs were incubated with 100 nM LysoTracker Red DND-99 (L7528, Thermo Fisher Scientific, Waltham, MA) for 10 min followed by three PBS washes prior to treatment with or without 100 nM concanamycin A. Cells were then imaged by confocal microscopy. Fluorescence confocal micrographs were acquired using the Ultraview Vox spinning-disk confocal system (PerkinElmer, Waltham, MA) equipped with a Yokogawa CSU-X1 spinning-disk head and an electron-multiplying charge-coupled device camera (Hamamatsu C9100-13) coupled to a Nikon Ti-E microscope equipped with a CFI Plan Apo VC ×60 oil objective. Z stacks (0.5-μm steps) were acquired with a Piezo z stack drive controlled by a nano drive (Mad City Lab, Madison, WI).

The following antibodies were used for immunofluorescence (IF): anti-E-cadherin (1:100; Cell Signaling), anti-β-catenin (1:100; Sigma-Aldrich), anti-Lamp1 (1:100; BD Biosciences), Alexa Fluor 568 goat anti-mouse secondary (1:500; Life Technologies), and Alexa Fluor 488 goat anti-rabbit secondary (1:500; Life Technologies). IF was performed as described previously (Overholtzer et al., 2007 (link)). Briefly, cells were cultured on glass-bottom dishes (P35G-1.5-20-C; MatTek) and were fixed in 1:1 methanol/acetone for 5 min at −20°C, followed by three 5-min PBS washes and blocking in 5% BSA, 100 mM glycine in PBS for 1 hr, followed by incubation with primary antibodies at 4°C overnight. Samples were then incubated with secondary antibodies and counterstained with DAPI (1:1,000; Life Technologies). Confocal microscopy was performed with the Ultraview Vox spinning-disk confocal system (PerkinElmer) equipped with a Yokogawa CSU-X1 spinning-disk head and an electron-multiplying charge-coupled device camera (Hamamatsu C9100-13) coupled to a Nikon Ti-E microscope; image analysis was performed using Volocity software (PerkinElmer).