Powergen 700

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).

Testes in 5 g batches were placed in a 50 mL culture tube containing 25 mL of Reagent B (50 mM Tris-HCl, pH 7.4, 0.25 M sucrose and 2.5 mL of reagent A), where reagent A is 1 μg/mL antipain, 1 μg/mL leupeptin, 10 μg/mL aprotinin, 13.2 μg/mL 4-amidinophenyl-methanesulfonyl fluoride hydrochloride (APMSF) and 250 mM KCl. The testes were minced with scissors and then blended by 10 s bursts repeated 2−3 times on Power Gen 700 (Fisher Scientific, Hampton, NH, USA) at setting 5–6, while on ice. The homogenate was centrifuged at 7500 rpm for 10 min at 4 °C using a Beckman SW28 rotor (5660 g). The resulting supernatant was collected and centrifuged at 23,500 rpm for 1 h at 4 °C in a Beckman SW40 rotor. The supernatant was discarded and the pellet containing the microsomes was suspended in 600 μL of reagent D (66% (v/v) reagent C, 10 mM DTT, 8 mM EDTA, 1 mM UDP-Glu and 1% CHAPSO) and dispersed by passage through a 25-gauge needle followed by an insulin needle. (15 mL of reagent C contained 0.17% N-laurosarcosine, 75 mM HEPES, pH 7.4, 30% glycerol, 0.03% NaN₃, 1.5 mM EDTA, 2.25 mL of reagent A, and 3 mM DTT.) The microsome suspension was stored as 100 μL aliquots in microcentrifuge tubes, flash frozen in liquid nitrogen for 1−2 min, stored at −80 °C, and used as needed.