Isolation and Preparation of Lysosomal Extracts from Dendritic Cells

DC lysosomal extracts were obtained as previously described using the lysosome isolation kit (Sigma-Aldrich, St. Louis, MO)^{26 (link),36 (link)}. Briefly, DCs were lysed using 1X extraction buffer (Sigma-Aldrich) followed by homogenization with a PowerGen 700 homogenizer (Fisher Scientific, Pittsburgh, PA) using the 7 × 110 mm homogenizer tip passed through the cells 15–20 times to disrupt 75–80% of the cells. The homogenized cells were then centrifuged at 1000 × g for 10 min to remove intact cells and cellular debris. The first supernatant was removed and centrifuged at 20,000×g for 20 min to pellet lysosomes. Lysosome purity was verified by flow cytometry for lysosomal marker LAMP1 (BD Biosciences, San Jose, CA), which showed > 90% pure lysosomes from this procedure. The pellet containing lysosomes was resuspended and sonicated for 20 s at 40% power on a Model 300 VT Ultrasonic Homogenizer (BioLogics, Inc., Manassas, VA), resulting in lysosome extract (600 mg/ml).