Flowjo apoptosis analysis11/9/2023 It relies on the property of cells to lose membrane asymmetry in the early phases of apoptosis. Proceed with the FITC Annexin V Staining Protocol to measure apoptosis.įITC Annexin V is used to quantitatively determine the percentage of cells within a population that are actively undergoing apoptosis. Prepare Camptothecin stock solution (Sigma-Aldrich Cat. The following protocol is provided as an illustration on how FITC Annexin V may be used on a cell line (Jurkat).ġ. Methods for utilizing Annexin V for flow cytometry on adherent cell types, however, have been previously reported (Casiola-Rosen et al. Investigators should note that FITC Annexin V flow cytometric analysis on adherent cell types (e.g HeLa, NIH 3T3, etc.) is not routinely tested as specific membrane damage may occur during cell detachment or harvesting. FITC Annexin V binding is calcium dependent and defined calcium and salt concentrations are required for optimal staining as described in the FITC Annexin V Staining Protocol. In contrast, a single observation indicating that cells are both FITC Annexin V and PI positive, in of itself, reveals less information about the process by which the cells underwent their demise.įITC Annexin V is a sensitive probe for identifying apoptotic cells, binding to negatively charged phospholipid surfaces (Kd of ~5 x 10^-2) with a higher affinity for phosphatidylserine (PS) than most other phospholipids. The movement of cells through these three stages suggests apoptosis. However, when apoptosis is measured over time, cells can be often tracked from FITC Annexin V and PI negative (viable, or no measurable apoptosis), to FITC Annexin V positive and PI negative (early apoptosis, membrane integrity is present) and finally to FITC Annexin V and PI positive (end stage apoptosis and death). This assay does not distinguish between cells that have undergone apoptotic death versus those that have died as a result of a necrotic pathway because in either case, the dead cells will stain with both FITC Annexin V and PI. For example, cells that are considered viable are FITC Annexin V and PI negative cells that are in early apoptosis are FITC Annexin V positive and PI negative and cells that are in late apoptosis or already dead are are both FITC Annexin V and PI positive. Viable cells with intact membranes exclude PI, wheras the membranes of dead and damaged cells are permeable to PI. Therefore, staining with FITC Annexin V is typically used in conjunction with a vital dye such as propidium iodide (PI) or 7-Amino-Actinomycin (7-AAD) to allow the investigator to identify early apoptotic cells (PI negative, FITC Annexin V positive). Since externalization of PS occurs in the earlier stages of apoptosis, FITC Annexin V staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation.įITC Annexin V staining precedes the loss of membrane integrity which accompanies the latest stages of cell death resulting from either apoptotic or necrotic processes. This format retains its high affinity for PS and thus serves as a sensitive probe for flow cytometric analysis of cells that are undergoing apoptosis. Annexin V may be conjugated to fluorochromes including FITC. Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding protein that has a high affinity for PS, and binds to cells with exposed PS. ![]() In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Loss of plasma membrane is one of the earliest features. The apoptotic program is characterized by certain morphologic features, including loss of plasma membrane asymmetry and attachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. Apoptosis is a normal physiologic process which occurs during embryonic development as well as in maintenence of tissue homeostasis.
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