Detect Cell Death: Apoptosis, Necrosis & Ferroptosis DOJINDO LABORATORIES

Science Note

[Apr. 1, 2025] Previous Science Note

Extracellular ATP Acts as Messenger in Immunity and Cancer

Extracellular ATP, released from dying or stressed cells, acts as a key damage-associated molecular pattern (DAMP) that signals immune cells and neighboring cells via purinergic receptors such as P2X7 and P2Y2. This Science Note introduces recent advances in our understanding of how extracellular ATP influences the immune system and the tumor microenvironment.

Colon tumour cell death causes mTOR dependence by paracrine P2X4 stimulation (Nature, 2022)

Colon tumor cells undergoing chemotherapy-induced cell death release extracellular ATP, which acts on neighboring tumor cells to activate mTOR signaling via P2X4 and promote their survival.

Highlighted technique: This study used Lgr5-EGFP-DTR(+) mice, which allow selective induction of apoptosis in Lgr5-positive (stem) cells by diphtheria toxin. Colon tumor organoids derived from these mice enabled targeted ablation of Lgr5-positive tumor cells, allowing analysis of the impact of their death on neighboring Lgr5-negative cells.

Related technique Extracellular ATP Assay, Annexin V Apoptosis Plate Assay

Cancer cells dying from ferroptosis impede dendritic cell-mediated anti-tumor immunity (Nature Communications, 2022)

To study the immune effects of ferroptosis cells, the researchers divided ferroptosis into initial, intermediate, and terminal phases, defined by lipid peroxidation, ATP release, and HMGB1 release with membrane rupture. Unlike apoptotic cells, ferroptosis cells fail to activate immunity through extracellular ATP and other signals, particularly in the initial phase, where they suppress dendritic cell maturation and phagocytosis.

Highlighted technique: This study established a model to precisely control cell death by inducing GPX4 knockdown, which triggers ferroptosis, and temporarily blocking ferroptosis by adding ferrostatin-1. Removal of ferrostatin-1 triggers synchronised ferroptosis, allowing the process of ferroptosis to be precisely controlled.

Related technique Intracellular Iron detection, Lipid Peroxide Detection

P2RX7 Enhances Tumor Control by CD8⁺ T Cells in Adoptive Cell Therapy (Cancer Immunology Research, 2022)

In melanoma, extracellular ATP enhances the antitumor activity of CD8⁺ T cells by maintaining their mitochondrial function through the purinergic receptor P2RX7.

Highlighted technique: Adoptive cell therapy (ACT) is a cancer treatment in which ex vivo activated immune cells are reinfused into the patient. As mitochondrial function is key to ACT efficacy, this study comprehensively evaluates T cell function using multiple parameters, including mitochondrial mass, mitochondrial ROS, oxygen consumption rate and extracellular acidification rate.

Related Techniques (click to open/close)

Target	Kit & Probes
Apoptosis detection in multiple samples	Annexin V Apoptosis Plate Assay Kit
Intracellular / mitochondrial ferrous ion (Fe²⁺) detection	FerroOrange, Mito-FerroGreen
Intracellular / mitochondrial lipid peroxidation detection	Liperfluo, MitoPeDPP
Mitochondrial membrane potential detection	JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
Mitochondrial superoxide detection	MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
Total ROS detection	Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA
Cell proliferation/ cytotoxicity assay	Cell Counting Kit-8 and Cytotoxicity LDH Assay Kit-WST

Application Note I (click to open/close)
> Apoptosis Inhibition Enhances ATP Release

Jurkat cells were treated with or without Z-VAD, an apoptosis inhibitor, and then treated with staurosporine. After treatment, changes in extracellular ATP release, cell viability and extracellular LDH release were assessed over time. The results showed that cell death was inhibited by Z-VAD, but extracellular ATP released during the initial phase of apoptosis increased over time.

＜Product in use＞
Extracelluar ATP
Extracellular ATP Assay Kit-Luminescence

LDH release
Cytotoxicity LDH Assay Kit-WST

Cell viability
Cell Counting Kit-8

Application Note II (click to open/close)
> Ferroptosis Induction and ATP Release Profile

Changes in extracellular ATP release, cell viability, extracellular LDH release, phosphatidylserine, and intracellular Fe²⁺ were evaluated in HeLa cells treated with various concentrations of Erastin, a ferroptosis inducer, for 24 hours. The results showed that cell viability decreased and extracellular ATP release and extracellular LDH increased in cells treated with Erastin concentration of 25 μmol/l, indicating that cell death was induced under high concentration conditions. Interestingly, the increase in extracellular ATP in the early phase of stimulation, which was observed with the apoptosis inducer Staurosporine, was not observed with Erastin (See Experimental Example: Evaluation using Staurosporine-treated Cells.). Although the apoptosis-related marker phosphatidylserine was not significantly altered by Erastin treatment at any concentration. The amount of intracellular Fe²⁺, a ferroptosis-related marker, was significantly increased under the low-concentration treatment condition, indicating that it tends to increase before actual cell death occurs.

＜Product in use＞
Extracellular ATP:
Extracellular ATP Assay Kit-Luminescence

Extracellular LDH:
Cytotoxicity LDH Assay Kit-WST

Cell viability: Cell Counting Kit-8

Phosphatidylserine:
Annexin V Apoptosis Plate Assay Kit

Intracellular Fe²⁺: FerroOrange

Topics

What is Cell Death?
Cell death detection methods
Multiple cell death indicators can be measured in combination with Dojindo's kits!
Experimental Example: Changes in various indicators of cell death induced by drugs
Experimental Example: Evaluation using apoptosis inhibitors
Experimental Example: Evaluation using Erastin-treated cells

What is Cell Death?

Cell death is a fundamental biological process essential for tissue homeostasis and stress response. Traditionally, programmed cell death, known as apoptosis, has been recognized as a key mechanism, while necrosis has been considered an unregulated response to injury. However, recent research has identified additional forms of programmed cell death, including ferroptosis, driven by iron-dependent lipid peroxidation, and autophagy-dependent cell death, resulting from excessive self-digestion of cellular components. Dysregulation of these pathways contributes to various cell death diseases, such as cancer, neurodegenerative diseases, and ischemic injury, where either evasion or excessive activation of cell death disrupts normal function. Understanding these mechanisms provides new therapeutic insights for cancer and other cell death-related diseases.

Cell death detection methods

Cell viability /Cytotoxicity Assay

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
	Extracellular ATP Assay	Extracellular ATP, known as damage-associated molecular patterns (DAMPs), are released from stressed or activated cells.	Extracellular ATP Assay Kit-Luminescence
	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

Category	Detection method	Principle	Dojindo products
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
	ADP/ATP Ratio Assay	It is known that ATP increases from ADP during the apoptotic process.	ADP/ATP Ratio Assay Kit-Luminescence
	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

Category	Detection method	Principle	Dojindo products
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

Category	Detection method	Principle	Dojindo products
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	-

Autophagy-dependent cell death

Category	Detection method	Principle	Dojindo products
Autophagy-dependent cell death	Autophagosome & Autolysosome Detection	Non-apoptotic autophagy-dependent cell death shows autophagic vacuolar structures.	Autophagy Flux Assay Kit Ref: Mori, H., et al., Mol. Metab, 2024

Multiple cell death indicators can be measured in combination with Dojindo's kits!

Same sample can be evaluated with multiple indicators.
By using cells and supernatant to measure each with different indicators, a more detailed analysis of cell death can be achieved.

Experimental example: 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.

[Products in use]
Extracellular LDH : Cytotoxicity LDH Assay Kit-WST (Product code: CK12)
Phosphatidylserine: Annexin V Apoptosis Plate Assay Kit（Product code: AD12）
Cell viability : Cell Counting Kit-8 (Product code: CK04)
Intracellular Fe²⁺ : FerroOrange (Product cose: F374) *Normalized with Hoechst 33342 fluorescence intensity
Lipid peroxidation : Lipid Peroxidation Probe -BDP 581/591 C11- (Product code: L267)

[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

Experimental Example: Evaluation using apoptosis inhibitors

Jurkat cells were treated with or without Z-VAD, an apoptosis inhibitor, and then treated with staurosporine. After treatment, changes in extracellular ATP release, cell viability and extracellular LDH release were assessed over time. The results showed that cell death was inhibited by Z-VAD, but extracellular ATP released during the initial phase of apoptosis increased over time.

＜Product in use＞
Extracelluar ATP : Extracellular ATP Assay Kit-Luminescence （code：E299）
Extracellular LDH : Cytotoxicity LDH Assay Kit-WST（code：CK12）
Cell viability : Cell Counting Kit-8（code：CK04）

Experimental Example: Evaluation using Erastin-treated cells

Changes in extracellular ATP release, cell viability, extracellular LDH release, phosphatidylserine, and intracellular Fe2+ were evaluated in HeLa cells treated with various concentrations of Erastin, a ferotosis inducer, for 24 hours. The results showed that cell viability decreased and extracellular ATP release and extracellular LDH increased in cells treated with Erastin concentration of 25 μmol/l, indicating that cell death was induced under high concentration conditions. Interestingly, the increase in extracellular ATP in the early phase of stimulation, which was observed with the apoptosis inducer Staurosporine, was not observed with Erastin (See Experimental Example: Evaluation using apoptosis inhibitors.). Although the apoptosis-related marker phosphatidylserine was not significantly altered by Erastin treatment at any concentration. The amount of intracellular Fe2+, a ferroptosis-related marker, was significantly increased under the low-concentration treatment condition, indicating that it tends to increase before actual cell death occurs.

＜Product in use＞
Extracellular ATP : Extracellular ATP Assay Kit-Luminescence （code：E299）
Extracellular LDH : Cytotoxicity LDH Assay Kit-WST（code：CK12）
Cell viability : Cell Counting Kit-8（code：CK04）
Phosphatidylserine : Annexin V Apoptosis Plate Assay Kit（code：AD12）
Intracellular Fe²⁺ : FerroOrange（code：F374）