Nis elements basic research software version 4

IHMGECs were cultured under differentiating conditions in a 1:1 mixture of DMEM/F12 (Mediatech, Inc., Manassas, VA), supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Rockford, IL). Azithromycin (AZM, 10 μg/ml, Santa Cruz Biotechnology, Dallas, TX) was added to the culture medium as a positive control for differentiation.^{19 (link)} After exposure to T4O (0.1%, 0.01%, and 0.001%), DMSO or AZM for up to 5 days, cells were stained for lysosome numbers using LysoTracker Red DND-99 (50 nM; Thermo Fisher Scientific) and for neutral lipid accumulation with HCS LipidTOX™ Green neutral lipid stain (1:200, Thermo Fisher Scientific) according to reported methods^{20 (link)}. Slides were examined by using an Eclipse E800 fluorescent microscope and images were captured with NIS-Elements Basic Research software, version 4.2 (Nikon Instruments, Melville, NY).

Cells were then stained for lysosome accumulation using LysoTracker® blue DND-22 and for neutral lipid with HCS LipidTOX™ Green neutral lipid stain.[30 (link)] Slides were viewed using an Eclipse E800 fluorescent microscope and images captured with NIS-Elements Basic Research software, version 4.2 (Nikon Instruments, Melville, NY,).[29 (link), 31 (link)-34 (link)] Intensities were quantified using ImageJ (http://rsb.info.nih.gov/ij).