Autophagosomes and autolysosomes were spatially examined using a lentivector for expression of a tandem-tagged mCherry-EGFP-LC3BB tracing construct  and the puromycin selection gene , a generous gift of Dr. Ana Maria Cuervo (Albert Einstein School of Medicine, NYC). Lentiviral particles were produced by co-transfection of HEK293T of the tamden-tagget plasmid with plasmids psPAX2 and pCMV-VSV (Addgene, Cambridge, MA) in the presence of LipoD293 (SignaGen, Ijamsville, MD). Culture supernatants were harvested in two batches in the 24–96 post transfection intervals, the combined supernatants were sieved through 0.45 μm filters and the viral particles were gently pelleted by centrifugation at 30,000 x g for 5 h and resuspended in culture medium. One set of age matched POAG and XFS TF were transduced with this lentivector 24 h after replating in the presence of 6 μg/ml polybrene (Fisher). Stably transduced cells were selected by culture complementation with 2 ug/ml puromycin for 48 h. Cells were cultured in glass bottom plates in SSFM. After 24 h the cells were incubated with 1 μg/ml Hoechst 33342 for 5 min to label nuclei and blue, green and red fluorescence emissions were captured in a in the Leica SP5 DMI confocal microscope equipped with a stage that maintains temperature at 37°C and CO2 at 5%.
The Cyto-ID Autophagy Detection Kit (Enzo Life Sciences, Farmington, NY) was used to compare autophagic flux rates between the XFS and POAG derived TF lines. Human TFs were seeded at 10 K cells/cm2 in SSFM in 12 well plates and were either treated with 10uM chloroquine for 18 h, at 37°C or left untreated (control). Following incubation, cells were trypsinized, collected by centrifugation, washed and resuspended in Cyto-ID Green stain solution in the dark for 30min at 37°C. After pelleting cells were resuspended in ice cold FACS buffer, consisting of phenol red-free (4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (Hepes)-buffered DMEM/F12 complemented with 5% FBS and 1 μg/ml propidium iodide (PI) and were immediately analyzed in an Accuri6 flow cytometer (BD, Franklin Lakes, NJ). All the flow cytometry measurements excluded any PIhigh (dead) cells and also all cells whose side light scattering (SSC; a relative measure of cellular granularity) to forward light scattering (FSC; a relative measure of cell size) showed a marked upward deviation from the main ratio (SSChigh cells). Such a deviation is a proven indication of apoptosis at early stages, due to membrane crenation . The average fluorescence in each sample was normalized to the average FSC of the sample. Full paper
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