Guidelines for Regulated Cell Death Assays: A Systematic Summary, A Categorical Comparison, A Prospective

Abstract

Over the past few years, the field of regulated cell death continues to expand and novel mechanisms that orchestrate multiple regulated cell death pathways are being unveiled. Meanwhile, researchers are focused on targeting these regulated pathways which are closely associated with various diseases for diagnosis, treatment, and prognosis. However, the complexity of the mechanisms and the difficulties of distinguishing among various regulated types of cell death make it harder to carry out the work and delay its progression. Here, we provide a systematic guideline for the fundamental detection and distinction of the major regulated cell death pathways following morphological, biochemical, and functional perspectives. Moreover, a comprehensive evaluation of different assay methods is critically reviewed, helping researchers to make a reliable selection from among the cell death assays. Also, we highlight the recent events that have demonstrated some novel regulated cell death processes, including newly reported biomarkers (e.g., non-coding RNA, exosomes, and proteins) and detection techniques.

Keywords: biomarkers; clinical application; detecting methods; guidelines; regulated cell death.

FIGURE 1

Timeline of the mile stone of cell death research. Abbreviations: Bcl-2: B-cell leukemia/lymphoma-2, CED-9: Cell death abnormality gene 9, RIPK3: Receptor-interacting serine/threonine-protein kinase 3, MLKL: Mixed lineage kinase domain like pseudokinase, PCD: Programmed cell death, CD95: cluster of differentiation 95.

FIGURE 2

Morphological changes of regulated cell death. Different types of cell death inducing with various pathways present diverse morphological changes. (A) The formation of autophagosomes is the typical characteristic of autophagy. ATG12-ATG5-ATG16L1 complex and LC3-II contribute to the extension of the phagophore. When the autophagosome is completely formed, LC3-II will separate from the outer membrane, and the autophagosome fuses with the lysosome so that an autolysosome finally emerged. (B) As for apoptosis, in the early stage, the nuclear condensation starts with cell shrinkage. During later stages, nucleus breaks up and the plasma membrane bubbles with no rupture, naturally associating with no inflammation. Finally, it forms apoptotic bodies. (C) Being different from apoptosis, after cell swelling and large bubbling, pyroptosis has plasma membrane rupture in the final stage. (D) When it comes to necroptosis, the appearance of cell swelling often followed by organelles dilation, and the nucleus disintegrates late. In some cases, chromatin condensation also occurs. Finally, with the rupture of plasma, a massive inflammation in the tissue is triggered. (E) The morphological characteristics of ferroptosis, however, mainly reflects on the changes of mitochondria. Compared with organelles swelling in necroptosis, here in the first stage, the mitochondria become smaller and membrane densities are elevated, followed by mitochondria crista reduction and plasma membrane rupture.

FIGURE 3

A schematic diagram showing biomarkers involved in RCDs within depicting the cross-regulations among different pathways. The solid arrows indicate activating interactions while the T-shaped lines indicate inhibitory interactions.