Preparation of Lysosomes from HeLa Cells and Mouse Liver Kupffer Cells

genate. From mice sacrificed after fasting for 18 hr, livers were rapidly removed and washed free of blood with cold 0.5 M sucrose. Livers (1 g) were minced with scissors, suspended in 3 ml of 0.5 M sucrose and homogenized by gentle pumping inside a 5 ml syringe without needle about 13 times. By this treatment almost all the Kunlfer cells in the tissues were disrupted. Their nuclei remained intact but the hepatocytes were not disrupted. To ascertain this point, Kupffer cells were isolated by the method of Pisano et al. [3], almost completely free from hepatocytes. Kupffer cells collected from 2 g liver were washed with 0.5 M sucrose by centrifuging at 500•~g for 10 min, suspended in 3 ml of 0.5 M sucrose and homogenized by gentle pumping within a 5 ml syringe without a needle as described above. Lysosomes were isolated from both the liver homogenate and the Kupffer cell homogenate and compared. Their biochemical behaviors were found to be almost the same and the lysosomes isolated from the liver homogenate seemed to be derived from the Kupffer cells. Preparation of lysosomes from the cell homogenate. Each HeLa cell and mouse liver (Kupffer cell) homogenate was centrifuged at 500•~g for 5 min to remove unbroken cells and whole nuclei and then the supernatant was centrifuged at 2500•~g for 10 min. The supernatant (4 ml) was layered onto 2 M sucrose (0.5 ml) and centrifuged at 32 000•~g for 30 min in a RPS 40 rotor in a Hitachi ultracentrifuge. Lysosome fraction banding above the sucrose cushion was pooled with a pipette and suspended in 0.5 M sucrose. These lysosomes were stable at 37 C for 60 min and