Isolation and characterization of mammalian cells that are undergoing apoptosis by a bovine serum albumin density gradient

When cells are treated with cytotoxic agents, they enter apoptosis asynchronously to yield cells at various stages of cellular deterioration. This mixture makes it difficult to study the biochemical pathways leading to cell death. We have fractionated apoptotic mammalian cells in a simple discontinuous bovine serum albumin (BSA) density gradient centrifugation into five layers, each containing cells at different stages of apoptosis, (1) nonapoptotic, (2) undergoing apoptosis, and (3) mature apoptotic cells, as judged by light and electron microscopy of chromatin condensation and by the extent of DNA fragmentation. Modifications of apoptosis markers including c-Jun N-terminal kinase/stress-activated protein kinase and procaspase 3 cleavage were apparent in those cells that are undergoing apoptosis. Apoptosis-specific histone H2B phosphorylation was highly elevated and DNA fragmentation activity in the cytoplasm was observed in those cells that are undergoing apoptosis, but not much was observed in the cells of other fractions. Results show that apoptotic cells can be fractionated easily by the BSA gradient method, and this method will be invaluable for studying the biochemical processes that drive apoptosis.