2.5. Confocal Laser Scanning Microscopy (CLSM)

For CLSM, cells were grown on a coverslip in a six-well Petri dish, and the following fluorescent reactions were carried out:

A Premo™ Autophagy Sensor LC3B-GFP (Life Technologies, Carlsbad, CA, USA) was used to detect LC3 protein localization; cells were transduced following the protocol provided by the datasheet [3]. This fluorochrome allows the visualization of autophagosomes and autophagolysosomes, which can be detected through fluorescent green puncta.

AO (Invitrogen, CA1301, Waltham, MA, USA) is a pH-sensitive dye used to detect acidic vesicular organelle formation, and it is considered a well-proven staining technique to monitor lysosomal membrane stability, mainly through fluorescence microscopy [3]. AO at the highest concentrations stains DNA, while at the lowest concentrations, in an acid environment, AO emits red fluorescence with an intensity proportional to the degree of acidity and/or to the acidic compartment volume. When the lysosomal proton gradient is lost, leakage of AO into the cytosol can be detected as a concomitant increase in green cytosolic staining and a loss of red lysosomal staining.

Cells were washed and re-suspended in 0.5 mL of medium and then stained with 75 ng/mL of AO for 15 min at 37 °C.

JC-1 (5,5′,6,6′-tetrachloro1,1′,3,3′-tetramethylbenzimidazolylcarbocyanine iodide; Invitrogen, T3168) was used to monitor mitochondrial membrane potential. The ratio of green/red fluorescence is independent from mitochondrial density, size, and shape, and it only correlates to the mitochondrial membrane potential. Therefore, JC-1 dye monomers form red fluorescent “J-aggregates” in the presence of a high mitochondrial potential, while a low potential corresponds to green fluorescent “J-aggregates”.

C2C12 differentiated cells from different treatment groups were washed in PBS and incubated with JC-1 (1 μM) in DMEM at 37 °C for 20 min.

All samples were observed with a Leica TCS-SP5 CLSM [24] connected to a DMI 6000 CS Inverted Microscope (Leica Microsystems CMS GmbH); LC3 (Exc/Em = 488/522); AO (Exc 488 nm, emission 480–560 nm (monomers), and 650 nm (stacks)); JC-1 (Exc 488 nm, emission 530 nm for dye monomers, and 590 nm for J-aggregates). Densitometric analysis of LC3 or JC1 immunofluorescence was performed with ImageJ 1.54j Software (National Institutes of Health).