Lysotracker green dnd 26

zVAD-fmk (627610) was purchased from Calbiochem (Gibbstown, NJ, USA). Chloroquine (#14774) and LysoTracker Green DND-26 (#8783) were purchased from Cell Signaling (Danvers, MA, USA). 3-Methyladenine (3-MA) (M9281), Bafilomycin-A1 (B1793), Phorbol-12-myristate-13-acetate (PMA) (P8139), Rapamycin (R0395), All-trans retinoic acid (ATRA) (R2625) and arsenic trioxide (A1010) were purchased from Sigma (St Louis, MO, USA). Perifosine (S1037) was purchased from Selleck Chemicals (Houston, TX, USA).

The assays MitoTracker CMXRos (20 nM), LysoTracker Green DND-26 (50 nM), and ER-Tracker Blue-White DPX (500 nM) were prepared according to the manufacturer’s instructions (Cell Signaling Technology, Thermo Fisher Scientific). After 72 h of treatment, Mitochondria and ER were stained for 30 min at 37 °C, and slides were washed twice. Acidic lysosomes were stained prior to analysis. Images were taken using fluorescence microscopy (Leica DMI 4000B).

Mitochondria were stained with MitoTracker^® Red CMXRos (M7512 ThermoFisher, MT-R). Acidic organelles were stained by Lysotracker Green DND-26 (#8783 Cellsignal, LT-G). For Mitotracker and Lysotracker stainings, cells were incubated for 30 min with Mitotracker (50 nM) and Lysotracker (50 nM) in prewarmed, freshly prepared culture medium (50% total medium change). In MN grown in microfluidic chambers, mitochondria were labeled by addition of a lentivirus overexpressing MitoDsRed (Prots et al., 2018 (link)). To analyze mitochondrial membrane potential, JC-1 (T3168, Invitrogen) was added to the medium at a final concentration of 1 μg/ml (50% medium change to prevent detachment) and cells were recorded after 60 min. As a positive control of mitochondrial depolarization, CCCP (50 μM final concentration, diluted in ethanol) was added to the medium 30 min before imaging. Lysosomes were labeled using immunohistochemistry for Lamp1 (see below) on day 31 differentiated neuronal cultures. In the ImageJ/Fiji software, the Lamp1 channel was thresholded using the auto function and the area of Lamp1 was determined within the region of the cell soma. For the quantification of Lamp1 positive organelles (LPO), enlarged perinuclear Lamp1 organelles were manually counted on blindcoded slides.

Primary microglia or astrocytes were seeded in a two-well chambered coverglass (ThermoFisher Scientific, Waltham, MA, USA) at equal density. The cell media was removed from the wells, then the wells were washed twice with DPBS, and 1 mL of phagocytosis assay medium containing 8 µL of pHrodo Red-conjugated synaptosomes was added into each well. The chambered coverglasses were kept in the incubator with 5% CO₂ at 37 °C for 40 min, then the phagocytosis assay media was removed, and the wells were washed thrice with DPBS to remove any unbound pHrodo Red-conjugated synaptosomes. Then 800 µL of fresh phagocytosis assay medium containing lysotracker green DND-26 (1:20,000, Cell Signaling Technology, Danvers, MA, USA) was added into each well. After 6 h of incubation, the live imaging of cells was performed using a Nikon Eclipse Ti (Nikon, Tokyo, Japan).

The following Reagents and antibodies were used in this study: Dulbecco’s Modified Eagle Media: Nutrient Mixture F-12 (Hyclone, UT, USA); 1 × phosphate buffered saline (1 × PBS; Hyclone, UT, USA); Fetal bovine serum (ExCell Bio, Australian); Hydrogen peroxide (H₂O₂) (Chemistry Industry Co, Shandong, China); Cell Counting Kit-8 (Beyotime, Shanghai, China); ECL developer (Beyotime, Shanghai, China); caspase-1 inhibitor Ac-TYR-VAL-ALA-ASP-CMK (Ac-YVAD-CMK) (Cayman Chemical, MI, USA); lysosomal inhibitor bafilomycin-A1 (Aladdin, Shanghai, China); Primary antibodies: anti-NLRP3, anti-caspase-1, anti-IL-1β, anti-CTSD, and anti-GSDMD (Abcam, Cambridge, MA, USA); anti-keratan sulfate (5D4) (MD bioproducts, MN, USA); anti-GAPDH (Proteintect, IL, USA); anti-p62 and anti-LC3 (Cell Signaling Technology, MA, USA); TWEEN-20 (Solarbio, Beijing, China); HRP-conjugated secondary antibody (Zsbio, Beijing, China); LysoTracker Green DND-26 (Cell Signaling Technology, MA, USA).