Pfb fdglu

PFB-FDGlu GCase activity assay: Cells in 96-well plates were pre-loaded for 30 minutes with lysotracker deep red (1:20,000, Invitrogen). Media was then exchanged for FluoroBrite imaging media (Thermo) containing 25uM of PFB-FDGlu (Invitrogen) and cells were then imaged on the Opera Phenix every 15 minutes for 2 hours to monitor GCase activity. Using the Columbus software, lysotracker signal was used to identify cells for quantification of GCase substrate fluorescence, which is depicted as the mean fluorescence per cell.
DQ-BSA: Cells were pre-loaded with DQ-BSA (Invitrogen) for the indicated duration in standard culture media. Cells were then stained with lysotracker deep red (1:20,000, Invitrogen) for 30 minutes and media was exchanged for FluoroBrite and imaging was conducted on the Opera Phenix. Using the Columbus software, lysotracker signal was used to identify cells and DQ-BSA fluorescence intensity was measured.

HT1080 CRISPRi cells were transduced with sgRNA-expressing lentiviral vectors and selected with antibiotics. 9 days after sgRNA transduction and 1 day after final re-plating, cells were assayed for their lysosomal hydrolase activity by incubating with 0.2 ug/mL Hoechst 33342 and either 200 uM PFB-FDGlu (for glucosylceramidase; P11947, Invitrogen) or 33 uM C₁₂FDG (for beta galactosidase; I2904, Invitrogen) in Imaging Media for 1 hour at 37°C, before imaging on the Phenix imager (Perkin-Elmer) with a 63x objective in confocal mode.
Image analysis was performed using the Harmony software (Perkin-Elmer), where flat-field corrected images were segmented for nucleus and cytoplasm. Total fluorescence intensity for each cell was extracted, and each biological replicate (two per condition) was represented by the median of the per-cell fluorescence (MFI) from all segmented cells, relative to the Non-targeting controls, as log10 fold-change.

For the lysosomal activity assay, SOD1^+/A4V or SOD1^+/+ MN were plated on a 96w plate at a density of 60,000 cells/well and were incubated with Dextran Blue (1 mg/ml working concentration) for 24h. Next day, the specific inhibitor Bafilomycin A1, which disrupts lysosomal pH and reduces the lysosomal activity of Glucocerebrosidase (GCase), was added to the culture at 200nM working concentration, while control wells were treated with the vehicle DMSO. One hour later the Dextran Blue was washed out and the cells were pulsed with the fluorescent substrate of the enzyme GCase, PFB-FDGlu (P11947 Invitrogen) at 100 μg/ml working concentration for 1h. Following incubation, the cells were washed once with media and phenol-free NBM was added to the wells. The fluorescence of PFB-FDGlu (485/535nm excitation/emission) was measured with microplate reader (Spectramax Gemini Microplate Reader, Molecular Devices) every 0.5h for up to 3h and normalized to the lysosomal mass (Dextran Blue, ex = 400, em = 430). In order to evaluate the selective lysosomal activity for each type of MN, the fluorescence derived from control wells treated with DMSO was subtracted from the respective wells treated with Bafilomycin A1.