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

Abstract

Studies of cellular apoptosis have been significantly impacted since the introduction of 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 in plasma membrane integrity and permeability 1,2 . The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells 3 . PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5 . The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6 . In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases 7,8 , allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9 . Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment 10 . Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence 10 . Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (< 5% events with cytoplasmic PI staining).

Chandipura Virus Induces Neuronal Death through Fas-Mediated Extrinsic Apoptotic Pathway

“…By following the manufacturer's instruction, we stained cells with FITC-annexin V and propidium iodide. Data were acquired and analyzed using Cell Quest Pro Software in FACSCalibur (Becton, Dickinson, San Diego, CA, USA) (27).…”

Antiproliferative activity of chloroformic fractions from leaves and inflorescences of Ageratina gracilis

“…As it is known, PI has the ability to enter a cell when it loses the permeability of the membrane, indicating late apoptosis or necrosis, therefore, PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane (Rieger et al, 2011). In this respect, the results indicated that the chloroformic fractions from petroleum ether extract of inflorescences, and chloroformic fraction from ethanolic extract of leaves induced early pro-apoptotic response on A549 cancer cell lines (Fig.…”

Synthesis of Europium-doped Fluorapatite Nanorods and Their Multifunctional Biomedical Applications

“…Propidium iodide (PI) was used in conjunction with Annexin V to determine if the cells were viable, apoptotic, or necrotic from differences in the plasma membrane integrity and permeability. This approach is a universal and accurate method for detecting cell apoptosis [16]. As shown in (Fig 4A&4C), few apoptotic cells were detected in the untreated control.…”