Discriminating Between Apoptosis, Necrosis, Necroptosis, and Ferroptosis by Microscopy and Flow Cytometry

Abstract

AbstractApoptosis is a mode of programmed cell death that plays important roles in tissue sculpting during development, in the maintenance of tissue homeostasis in the adult, and in the eradication of injured or infected cells during pathological processes. Numerous physiological as well as pathological stimuli trigger apoptosis, such as engagement of plasma‐membrane‐associated Fas, TRAIL, or TNF receptors, growth factor deprivation, hypoxia, radiation, and exposure to diverse cytotoxic drugs. Apoptosis is coordinated by members of the caspase family of cysteine proteases, which, upon activation, trigger a series of dramatic morphological and biochemical changes including retraction from the substratum, cell shrinkage, extensive and protracted plasma membrane blebbing, chromatin condensation, DNA hydrolysis, nuclear fragmentation, and proteolytic cleavage of numerous caspase substrates. These dramatic structural and biochemical alterations result not only in the controlled dismantling of the cell, but also in the rapid recognition and removal of apoptotic cells by phagocytes through the cell surface display of phagocytotic triggers such as phosphatidylserine. Necrosis, which is typically nonprogrammed or imposed upon the cell by overwhelming membrane or organelle damage, is characterized by high‐amplitude cell swelling, followed by rapid plasma membrane rupture and release of cellular contents into the extracellular space. Necrosis is often provoked by infectious agents or severe departure from physiological conditions due to toxins, temperature extremes, or physical injury. However, forms of programmed necrosis (necroptosis, pyroptosis, ferroptosis) can also occur in specific circumstances. Nonprogrammed and programmed necrosis can be distinguished from apoptosis by morphological features, based on the rapid uptake of vital dyes, and through the application of specific inhibitors of key molecules associated with the latter modes of cell death. This unit describes protocols for the measurement of apoptosis and necrosis and for distinguishing apoptosis from programmed as well as conventional necrosis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Analysis of cell morphology by phase‐contrast microscopyAlternative Protocol 1: Assessment of morphological changes using eosin‐methylene blue stainingAlternative Protocol 2: Analysis of nuclear morphology by fluorescence microscopySupport Protocol: Preparation of cytospinsBasic Protocol 2: Measurement of plasma membrane composition with annexin V and propidium iodideBasic Protocol 3: Measurement of DNA fragmentation by flow cytometryAlternative Protocol 3: Analysis of DNA fragmentation by the TUNEL assayBasic Protocol 4: Measurement of caspase activation by flow cytometryBasic Protocol 5: Discriminating between apoptosis, necrosis, necroptosis, and ferroptosis