Quantification of apoptotic cells by TUNEL assay

SK-N-SH were exposed to His-CATB [250 ng/mL] and all the treatments described above, diluted in plain EMEM. Cells were treated and incubated at 37 °C for 24 hours as described⁶. His-CATB/EMEM solution was pretreated with specific cathepsin B inhibitor CA-074 (Sigma-Aldrich, 10 µM) or monoclonal anti-cathepsin B, or anti-SAPC antibodies. During apoptosis, fragmented DNA exhibits green fluorescence upon TUNEL labeling. A minimum of three images were acquired for each condition for each donor. Green fluorescent nuclei were counted using ImageJ software (NIH) and divided by the total number of neurons (all DAPI-positive nuclei, blue), to obtain a percentage of apoptotic neurons. A dilution of 30% exosomes in EMEM and 25% MCM in EMEM were added. Negative controls included SK-N-SH exposed to His-CATB solution pre-treated with IgG1 isotype control, His-GAPDH [250 ng/mL] (as a Histidine-tagged protein negative control) added in EMEM, uninfected MCM and HIV + MCM, and untreated SK-N-SH incubated in plain EMEM. TUNEL assay positive control constitutes SK-N-SH treated with DNaseI to promote DNA fragmentation and positive green staining.