Detect Cell Death: Apoptosis, Necrosis & Ferroptosis DOJINDO LABORATORIES

What is Cell Death?

Cell death is a fundamental biological process that is essential for maintaining tissue homeostasis and responding to cellular stress. Traditionally, apoptosis, triggered by cellular signals leading to programmed cell death, and necrosis, caused by external injury or infection, have been considered the major types. Recent research has identified several other mechanisms, such as ferroptosis, which results from iron-dependent lipid peroxidation, and autophagy-dependent cell death, which results from excessive self-digestion of cellular components.

Science Note

Advancing Our Understanding of Cell Death Mechanisms [Feb. 4, 2025]

In recent years, apoptosis, along with other distinct types of cell death, such as ferroptosis and pyroptosis, have been identified, making the classification and pathways of cell death increasingly complex. This Science Note introduces a review of cell death pathways and highlights recent findings on the induction of apoptosis and the clearance of apoptotic cells.

Cell death Review Article
Kim Newton, et. al., Cell, 2024.
Frequently cited throughout 2024, this review provides a comprehensive overview of apoptosis, ferroptosis, and other cell death processes, as well as their associated diseases. It will be a helpful resource for researchers beginning to study cell death and seeking to understand its complex pathways.

Stem cells tightly regulate dead cell clearance to maintain tissue fitness
Katherine S. Stewart, et. al., Nature, 2024.
Stem cells are non-motile and non-professional phagocytes, yet they possess the ability to engulf apoptotic corpses.A commonly used method to detect phagocytosis, as described in this paper, is to label the engulfing and engulfed cells with different fluorophores and to detect double-positive cells.

Acute suppression of mitochondrial ATP production prevents apoptosis and provides an essential signal for NLRP3 inflammasome activation
Benedikt S. Saller, et. al., Immunity, 2025.
Apoptosis and NLRP3 inflammasome-mediated pyroptosis are distinct cell death pathways, but mitochondrial signals determine which pathway the cell follows. This paper describes the preparation methods for various primary cells, including PBMCs, thymocytes, microglia, and Kupffer cells, which will be useful to researchers performing immune cell experiments.

Related Techniques (click to open/close)

Target	Kit & Probes
Cell proliferation/ Cytotoxicity assay	Cell Counting Kit-8, Cytotoxicity LDH Assay Kit-WST
Apoptosis detection in multiple samples	Annexin V Apoptosis Plate Assay Kit　NEW
Ferroptosis Indicator: ferrous ion (Fe²⁺)	FerroOrange(intracellular), Mito-FerroGreen(mitochondria)
Ferroptosis Indicator: lipid peroxidation	Liperfluo(intracellular), MitoPeDPP(mitochondria)
Mitochondrial membrane potential detection	JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
Mitochondrial superoxide detection	MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
Oxygen consumption rate assay	Extracellular OCR Plate Assay Kit
Total ROS detection	Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA
Glycolysis/Oxidative phosphorylation Assay	Glycolysis/OXPHOS Assay Kit

Application Note (click to open/close)
> Changes in various indicators of cell death induced by drugs

HepG2 cells treated with the apoptosis-inducing agent staurosporine or the ferroptosis-inducing agents Erastin and RSL3. After treatment, extracellular LDH, phosphatidylserine, cell viability, intracellular Fe²⁺ and lipid peroxidation were determined.

The results showed that apoptosis-induced cells treated with staurosporine showed an increase in phosphatidylserine, a decrease in cell viability and an increase in extracellular LDH, indicating that cell death had occurred. On the other hand, intracellular Fe²⁺, an indicator of ferroptosis, remained unchanged. In cells treated with Erastin, a ferroptosis inducer, intracellular Fe²⁺ increased and cell viability decreased, but extracellular LDH and lipid peroxidation (lipid peroxidation: decrease in red fluorescence and increase in green fluorescence) did not increase. In cells in which ferroptosis was more strongly induced by co-treatment with RSL3 in addition to Erastin, increased intracellular Fe²⁺ and lipid peroxidation were observed. Moreover, decreased cell viability and increased dead cells were detected. Meanwhile, phosphatidylserine showed a lower rate of increase during ferroptosis induction compared to apoptosis-induced cells. These results suggest that cell death can be distinguished by evaluating a combination of cell death indicators.

[Experimental conditions]
Cell type: HepG2 cell(2×10⁴ cells/well)
Drugs: Staurosporin(5 μmol/l), Erastin(25 µmol/l), Erastin+RSL3(both 25 µmol/l) *Diluted in serum-free medium

Cell death detection methods

Category	Detection method	Principle	Dojindo products
Cell viability /Cytotoxicity Assay	WST assay	The WST-8 is reduced by dehydrogenase activities in cells to give a yellow-color formazan dye, which is soluble in culture media.	Cell counting Kit-8
	Calcein assay	The amount of the fluorescent dye, calcein, hydrolyzed by esterases in cells is directly proportional to the number of viable cells.	Cell counting kit-F
	MTT Assay	MTT can pass through a cell membrane and is reduced by mitochondria to form a purple color formazan dye.	MTT
	LDH detection assay	LDH leaks from dead cells after cell membrane breakdown.	Cytotoxicity LDH Assay Kit-WST
	Live / dead cell staining	Stain cells with calcein, which stains live cells, and PI, which stains dead cells, to determine whether cells are live or dead.	-Cellstain- Double Staining Kit
Apoptosis	Annexin V binding assay	Annexin V detection of phosphatidylserine, a hallmark of apoptosisAccurate plate assay without the need for washing.	Annexin V Apoptosis Plate Assay Kit
	DNA fragmentation	DNA fragmentation occurs due to the activation of the caspase cascade.	-
	p53 activity detection	p53 promotes apoptosis through transcription-dependent and independent mechanisms.	-
	Caspase activity	Apoptosis transitions to the execution phase, which is triggered by caspase-3 activation.	-
	Cytochrome c release	Release of cytochrome c form mitochondria to cytosol is considered sign of apoptosis.	-
Necrosis	LDH detection assay	LDH leaks from dead cells after cell membrane breakdown.	Cytotoxicity LDH Assay Kit-WST
	Nucleus staining	Non-membrane permeable nuclear staining dyes accumulate in the nucleus of cells with damaged cell membranes.	-Cellstain- DAPI solution -Cellstain- PI solution
	RIP/RIP3/MLKL	Marker proteins involved in the necrosis pathway	-
Ferroptosis	Ferrous ion detection	The dye stains Fe²⁺ that induces lipid peroxidation, a key factor in ferroptosis.	FerroOrange
	Mitochondrial ferrous ion detection	The dye stains mitochondrial Fe²⁺, which is a key factor in ferroptosis.	Mito-FerroGreen
	Lipid peroxides detection	The dye Specifically detects lipid peroxides, an inducer of ferroptosis.	Liperfluo
	GSH/GSSH	Glutathione is present in cells in either the reduced form (GSH) or the oxidized form (GSSG) and protects cells from oxidative stress.	GSSG/GSH Quantification Kit
	Cystine detection	Cystine is imported into cells via the xCT transporter and reduced to cysteine, an important precursor for glutathione (GSH) synthesis.	Cystine Uptake Assay Kit
	GPX4 / SCL7A11 / Ferritin / NRF2	Ferroptosis protein markers	-