Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

摘要: Studies of cellular apoptosis have been significantly impacted since the introduction flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences plasma membrane integrity and permeability1,2. The V/ PI protocol a commonly approach for studying apoptotic cells3. more often than other nuclear stains because it economical, stable good indicator cell viability, based on its capacity exclude dye living 4,5. ability enter dependent upon permeability membrane; does not stain live early due presence an intact 1,2,6. In late cells, membranes decreases7,8, allowing pass membranes, intercalate into nucleic acids, display red fluorescence 1,2,9. Unfortunately, we find that conventional protocols lead significant number false positive events (up 40%), which associated staining RNA within cytoplasmic compartment10. Primary lines broad range animal models affected, large (nuclear: ratios <0.5) showing highest occurrence10. Herein, demonstrate modified method provides improvement assessment death compared This takes advantage changes during fixing promote entry RNase A following staining. Both timing concentration optimized removal RNA. result over (< 5% staining).