100mg tissue was rinsed with 1XPBS, homogenized in 1 ml of 1X PBS and stored overnight at -20°C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 x g, 2 - 8°C. The supernate was assayed and removed immediately. Alternatively, aliquot and store samples at -20°C or -80°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles