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Abstract

The objective of this study was to investigate the sensitivity, specificity and reproducibility of some frequently used apoptosis assays. The degree of apoptosis was tested in two T-lymphoblastoid cell lines, HSB and Jurkat, in which apoptosis was induced by ionizing radiation. HSB and Jurkat samples were taken before, and 0, 2, 4, 6, 8 and 24 h after irradiation with 6 and 10 Gray, or with 10 and 14 Gray, respectively. Four frequently used flow cytometric techniques were evaluated: (i) Annexin V/Propidium Iodide assay, detecting the translocation of phosphatidylserine to the outer leaflet of the plasma membrane, simultaneously with preservation of the membrane integrity; (ii) Terminal deoxynucleotidyl Transferase (TdT) Uridine triphosphate (UTP) nick end labelling (TUNEL), revealing the presence of DNA strand breaks; (iii) DNA-flow cytometry, measuring DNA-stainability (DNA-fragmentation assay) and (iv) Phycoerythrin-labelled (PE) Apo2.7-assay, a monoclonal antibody against 7A6 antigen, a protein, which becomes exposed upon the mitochondrial membrane during apoptosis. As a general standard for identifying that apoptosis had occurred, the cells were assessed for the presence of DNA-laddering on agar gel electrophoresis and by demonstration of characteristic cell morphology. Results were as follows: Fluorescein Isothiocyanate (FITC)-labelled Annexin V/Propidium iodide flow cytometry appeared to be the most sensitive, the most specific and the most user-friendly test for measurement of apoptosis of cells in culture conditions in suspension. The expression of 7A6 antigen on the mitochondrial membrane appeared to be not specific for apoptotic cell death.

Mitochondrial Aberrations in Mucolipidosis Type IV

“…There was some degree of variability to the exact values yielded by these assays, which has been previously observed (52,53). Nonetheless, the three assays demonstrated the same trend: under control conditions, the percentage of MLIV cells undergoing apoptosis was not statistically different from MLIV-control fibroblasts (Fig.…”

Blebs produced by actin–myosin contraction during apoptosis release damage-associated molecular pattern proteins before secondary necrosis occurs

“…17 Despite the general application of this binary classification, we observed that apoptotic blebs in NIH3T3 fibroblasts treated with tumor necrosis factor α (TNF α ) plus cycloheximide (CHX) can permit PI penetration while the cell body continues to exclude the nucleic acid stain (Figure 1a). Time-lapse microscopy revealed that shortly after blebbing onset (∼4 h after TNF α /CHX treatment), newly formed blebs and apoptotic bodies (small membrane-encapsulated subcellular particles that dissociate from blebbing cells) appear that do not exclude PI (Figure 1b, from 4 : 14 : 30, examples indicated by white, blue, yellow or red arrows).…”

How apoptotic cells aid in the removal of their own cold dead bodies

“…One of the key features of apoptotic cells that allows for the rapid and stealthy removal of cellular fragments is a stable intact membrane (detectable by the exclusion of impermeable dyes such as propidium iodide) that prevents release of intracellular proteins and consequent immunological activation. 48 This is in contrast to necrotic cell death, wherein cells inappropriately lyse and release their intracellular contents leading to rapid pro-inflammatory responses. 45, 49 Secondarily necrotic cells release ‘alarmins', of which high-mobility group protein B1 (HMGB1) is the archetype, that are recognized as danger signals and provoke innate immune cells into a pro-inflammatory state (Figure 3).…”