For LysoTracker labelling, cells were incubated at 37°C with 50 nM of LysoTracker (Molecular Probes) during 2 h, and subsequently washed, before fixing.
For LDL incorporation cells were cultured in FITC-conjugated acetylated LDL (ac-LDL, 1∶1000, L23380, Invitrogen – Molecular Probes) during 4 h before fixation.
Presynaptic vesicles were labeled by exposure to FM1-43 (15 µMolecularlar Probes, USA) during a high-K+ depolarization for 5 min and immediately washed, as previously described [39], [40].
Alternatively, ROS generation was assessed in astrocytes cultured on coverslips with the fluorogenic marker carboxy-H 2DCFDA (Molecular Probes, USA) during 30 min/37ºC, protected from the light.
In some experiments, myocytes were incubated with 500 nM MitoTracker Red 580 (Molecular Probes, Eugene, OR) during this period.
F-actin was stained by incubating with 0.165 mM rhodamine-conjugated phalloidin (Molecular Probes, Eugene OR, USA) during 20 minutes in the dark at room temperature.
To measure intracellular ROS, treated and untreated cells were loaded with 10 μM carboxi-H2DCFDA probe (Molecular Probes; Europe BV, Leiden, The Netherlands) during the last 30 min of treatment.
Invasion was quantified by exposing the cells in the lower compartment to 5 μ M calcein-AM (Molecular Probes, Leiden, the Netherlands) at 37°C during the final 30 min of the assay.
The filamentous actin dye, phalloidin (1 200, AlexaFluor 488, Molecular Probes), was applied for 30 min during the secondary antibody incubation.
TO-PRO-3 (Molecular Probes) was used (1∶1000) to stain DNA during 90 min, at room temperature.
We used CM-H2DCFDA (Molecular Probes, Invitrogen) to measure the intracellular ROS during the reoxygenation (1 6 hr) according to the manufacturer's protocol with a few modifications.
