详细信息:Millipore’s FlowCellect™MitoDamage Kit includes MitoSense Red, a fluorescent cationic dye that accumulates in the mitochondria, and is responsive to mitochondrial potential changes. Annexin V conjugated to a green sensitive dye, CF488A, which binds to phosphatidylserine (PS) on the surface of apoptotic cells. And the cell impermeant DNA intercalator 7-Aminoactinomycin-D (7-AAD), a dead cell dye. The simultaneous use of the reagents allows researchers to obtain information on early, mid and late apoptosis in one simple assay.
MitoSense Red is excitable by a red laser and fluoresces maximally at 650 nm (Red2 fluorescence on the guava easyCyte 8HT). Uninduced cells with intact mitochondrial membrane potential demonstrate high Red2 fluorescence while cells which have impaired mitochondrial membrane potential depict lower Red2 fluorescence. Annexin V is calcium-dependent phospholipid binding protein with high affinity for PS, a membrane component normally localized to the internal face of the cell membrane. It is conjugated to CF488A which is excited by a 488 nm laser and emits at 525 nm. Control cells depict no green fluorescence while apoptotic cells exhibit positive green fluorescence. The 7-AAD dye included in the kit monitors cell membrane permeability changes typically observed later in apoptosis as well as necrotic cell death. 7-AAD is excluded from live, healthy cells as well as early apoptotic cells and these cells have low red fluorescence. The kit can thus distinguish multiple populations: 1) Live cells with intact mitochondrial membrane 2) Cells with dissipated membrane potential but no Annexin V or 7AAD staining 3) Early apoptotic cells with dissipated membrane potential and Annexin V binding 4) Late Apoptotic Cells or dead cells with dissipated membrane potentials. The kit thereby provides a complete picture of mitochondrial health and cell health and allows the correlation of mitochondrial damage to cell health. The entire assay can be performed in less than30 minutes once cellular samples are ready
