Apoptosis, or programmed cell death is a well-documented phenomenon that has been observed in many cell systems. Observed features of apoptosis include decreased cell size, chromatin condensation, and nuclear fragmentation due to endonuclease cleavage of DNA.
Changes in the phospholipid bilayers of cell membranes are also observed early in the apoptosis process. The phosphotidylserine (PS) component of the phospholipid bilayers are externalized and can be detected by fluorescence labeling. Annexin V is a member of the annexin family of calcium-dependent phospholipid-binding proteins. Annexin V has a high affinity for PS-containing phospholipid bilayers.
Staining with FITC-conjugated annexin V and propidium iodide (PI) can identify subpopulations of cells with membrane changes and the associated loss of membrane integrity. When analyzed using the LSC, cells can be relocated by CompuSort to observe the localization of Annexin V and PI in these subpopulations.
Lymphocytes were prepared using the Annexin V-FITC Apoptosis Detection Kit (Genzyme Corp., Cambridge, MA) according to manufacturer's directions, with the exception that the sample volumes and cell concentrations were adapted to the LSC system. Only 10 µl of each specimen at a concentration of 4 X 106 was needed for analysis on the LSC.
The LSC utilized the argon laser operating at 5 mW. The cell detection threshold was set to select single cells based on forward angle light scatter displayed in a dot plot of cell area vs. forward scatter integral. Cells from the region determined to be single lymphocytes were electronically gated to a dot plot of FITC integral vs. PI integral as shown above.
Cells were gated based upon Annexin V binding and three levels of PI staining. CompuSort found the cells from each region of the dot plot by extracting the X and Y position data for each cell from the .fcs file and positioning the microscope stage to present each cell for image capture.
(Data shown are from the Laboratory of Immunology, Department of Pathology, Tufts University, Boston, MA.)
See also APOSCAN: Specifically configured LSC for analyzing apoptotic samples
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