Lysotracker deep red

Cells were seeded the day before the assay. The next day, cells were incubated with Lysotracker DeepRed (Thermo Fisher Scientific L12492) according to the manufacturer protocol. The cells were examined with Nikon Ti-Eclipse microscope and analyzed with Volocity 6.3 software (Perkin Elmer). For subsequent analysis, cells were then fixed with paraformaldehyde 3% for 10 minutes at room temperature and nuclei were stained with DAPI (Molecular Probes NucBlue Live ReadyProbes Reagent R37605) as manufacturer protocol, then washed and mounted on slides with Mowiol 20%. The cells were examined by confocal microscopy (Leica SP5) and analyzed with Volocity 6.3 software (Perkin Elmer). Lysotracker experiments were performed at least three times, in triplicate.

We used the marker LAMP1 (lysosomal-associated membrane protein 1) fused with RFP to detect lysosomes and endolysosomes, transducing either neurons or HEK293T cells with a baculoviral vector (C10597, Thermo-Fisher) for 48 h followed by confocal microscopy analysis of fixed cells. To track acidic lysosomes, cells were treated with LysoTracker^® Deep Red (L12492, Thermo-Fisher) at 66 nM concentration in the culture medium, and incubated at 5% CO₂ and 37 °C for 1 h. The cells were then washed 3 times with PBS prior to fixation and confocal imaging. To test the effect of alkalinization on lysosomes, the tau biosensor cells were treated with exosomes in culture media containing ammonium chloride (50 mM).

Approximately 2 ×
10⁵ A549 cells in cell culture medium (2 mL) were seeded
on a confocal dish and incubated overnight at 37 °C in a humidified
5% CO₂ atmosphere. After being rinsed with PBS, the cells
were incubated with 1 (4 μM) at 37 °C for
1 h. After that, the cells were stained with LysoTracker Deep Red
(Thermo Fisher Scientific, Inc., L12492) (0.1 μM for 30 min),
MitoTracker Red CMXRos (Thermo Fisher Scientific, Inc., M7512) (0.1
μM for 20 min), or ER-Tracker Red (Thermo Fisher Scientific,
Inc., E34250) (1 μM for 20 min) in a serum-free medium at 37
°C. The solutions were then removed, and the cells were rinsed
with PBS twice before being examined with a Leica TCS SP8 high-speed
confocal microscope equipped with a 488 nm laser, a 552 nm laser,
and a 638 nm laser. LysoTracker Deep Red was excited at 638nm, and
the fluorescence was monitored at 650–680 nm. MitoTracker Red
CMXRos and ER-Tracker Red were excited at 552 nm, and their fluorescence
was monitored at 590–620 nm. Compound 1 was excited
at 488 nm and its fluorescence was monitored at 500–600 nm.
The images were digitized and analyzed using a Leica Application Suite
X software.

Rat cardiomyoblast H9c2 cells (Sigma-Aldrich, 88092904) were cultured in high-glucose (4.5 g/L) Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich, D5796) with 10% Fetal Bovine Serum (FBS) and 1% streptomycin/penicillin (Sigma-Aldrich, P4333). For glucose deprivation and adaptation to galactose, the cells were grown in DMEM without glucose (Gibco, 11966–025) supplemented with 2 mM L-glutamine (Sigma-Aldrich, G7513) 1 mM sodium pyruvate (Sigma-Aldrich, S8636), 10 mM galactose (Sigma-Aldrich, G5388), 10% fetal bovine serum (Sigma-Aldrich, F7524) and 1% streptomycin-penicillin (Sigma-Aldrich, P4333). The cells were adapted to galactose for at least 7 days before the experiments. Stable H9c2 cells (see below) were grown in the same medium with the addition of 1 μg/ml of puromycin (InvivoGen, ant-pr-1). For hypoxic conditions, the cells were incubated at 0.3% O₂ for 2, 4, 6 or 16 h. For labeling of lysosomes, the cells were treated with 50 or 100 nM LysoTracker Deep Red (ThermoFisher Scientific, L12492) for 30–40 min or with LysoView 650 (Biotium, 70059). Cells were treated as indicated with 0.2 μM bafilomycin A₁ (BafA1 from Streptomyces griseus; Sigma-Aldrich, B1793) or 10 μg/ml pepstatin A (Sigma-Aldrich, P5318) and 10 μg/ml E64d (Sigma-Aldrich, E8640). All cell lines were maintained at 37°C and under 5% CO₂.

HEK293 cells (from Leibniz-Institut DSMZ, Braunschweig, Germany) were transfected with polyethyleneimine and 2 μg of plasmid DNA in serum-free medium. After 5 hours, complete medium was added. Cells were transfected with the following constructs: GFP (as control), SRF-VP16, SRF-VP16 ΔMADS (44 (link)), constitutively active MRTF-A (52 (link)), Poly-GA (75 (link)), SOD1^G93A (47 (link)), SOD1^A4V (45 (link)), or P62-GFP-mCherry (46 (link)). For the Poly-GA–lysotracker experiment, after overnight incubation, 1 μM Lysotracker Deep Red (Thermo Fisher Scientific) was added and incubated for 2 hours at 37°C. For qPCR experiments with HEK293 cells, RNA was extracted with TRIzol (Qiagen). For reverse transcription, 1 μg RNA was mixed with random primers dN6 (Biomers), incubated for 10 minutes at 70°C, and placed on ice. Then, the master mix (room temperature 5× buffer, Promega Biosciences), dNTPs (Genaxxon), Ribolock RNase Inhibitor (Thermo Fisher Scientific), and M-MLV RT RNase (Promega Biosciences) were added. After 10 minutes at room temperature, the mixture was incubated at 42°C for 45 minutes, at 99°C for 3 minutes, and, thereafter, on ice.