The release of microparticles and mitochondria from RAW 264.7 murine macrophage cells undergoing necroptotic cell death in vitro

Abstract

Microparticles (MPs) are small membrane-bound vesicles released from activated or dying cells. As shown previously, LPS stimulation of the RAW 264.7 macrophage cell line can induce MP release, with the caspase inhibitor Z-VAD increasing the extent of this process. Since combined treatment of cells with LPS and Z-VAD can induce necroptosis, we explored particle release during this form of cell death using flow cytometry to assess particle size, binding of annexin V and staining for DNA with propidium iodide (PI) and SYTO 13. The role of necroptosis was assessed by determining the effects of necrostatin, an inhibitor of RIP1, a kinase regulating this form of cell death. These studies demonstrated that, during necroptosis, RAW 264.7 cells release MPs that resemble those released from cells treated with staurosporine to induce apoptosis. The particles contained DNA as determined by binding of PI and SYTO 13, with PCR analysis demonstrating both chromosomal and mitochondrial DNA. The presence of mitochondria in the MP preparations was demonstrated by staining with MitoTracker Green. Flow cytometry indicated that purified mitochondria have properties of MPs. Together, these studies indicate that cells undergoing necroptosis can release MPs and that mitochondria can be components of MP preparations.