Detect Cell Death: Apoptosis, Necrosis & Ferroptosis DOJINDO LABORATORIES

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

Science Note

[May 6, 2025] Previous Science Note

Regulation of ATP Release in Cell Death

ATP release during cell death plays a central role in immune activation, but different cell death modalities either suppress, delay or enhance this release through different molecular mechanisms. This Science Note introduces recent studies that shed light on how different cell death pathways regulate ATP dynamics and influence immunological outcomes.

An unconventional autophagic pathway that inhibits ATP secretion during apoptotic cell death (Nature Communications, 2025)

Summary: While cell death typically involves the release of DAMPs such as ATP, which trigger immune responses, certain forms of apoptosis suppress this release. This study shows that ATP is sequestered via an unconventional autophagic pathway, thereby limiting its extracellular release and maintaining the immunosilent nature of apoptosis.

Highlighted technique: This study showed that blocking the BAK-dependent non-canonical autophagy pathway during apoptosis increased extracellular ATP and reduced ATP in apoptotic bodies. Co-culture with ATP and these apoptotic bodies enhanced IL-1β production by macrophages, suggesting that inhibition of this pathway promotes immune activation.

Related technique Apoptosis Plate Assay, Extracellular ATP Assay, Autophagy Flux Detection

Gasdermin D mediates a fast transient release of ATP after NLRP3 inflammasome activation before ninjurin 1-induced lytic cell death (Cell Reports, 2025)

Summary: Pyroptosis is a form of necrotic cell death induced by cleavage of gasdermin D (GSDMD) and subsequent pore formation by the N-terminal domain. This study shows that at an early stage of GSDMD-mediated plasma membrane permeabilization, ATP is transiently released prior to cell death and IL-1β secretion, acting as an early danger signal.

Highlighted technique: To investigate ATP release, the authors used GSDMD-deficient mice and found that extracellular ATP was not released upon NLRP3 activation without GSDMD. In contrast, inhibition of membrane rupture suppressed LDH but not ATP release, suggesting that ATP release is an early event independent of cell lysis.

Related technique Extracellular ATP Assay, Released LDH Assay

An inducible RIPK3-driven necroptotic system enhances cancer cell–based immunotherapy and ensures safety (J. Clin. Invest., 2024)

Summary: Some cancer therapies are exploring the use of genetically engineered cancer cells to stimulate the immune system in a safe and controlled manner. This study shows that activating a RIPK3-based cell death system in such cells triggers the release of ATP, which boosts the immune response and improves both the safety and efficacy of cancer cell-based treatments.

Highlighted technique: The researchers engineered cancer cells to express a drug-inducible RIPK3 construct and implanted these cells into the brains of mice. When the RIPK3 switch was activated in vivo, necroptosis was induced, leading to the release of ATP and DAMPs, which in turn stimulated anti-tumour immune responses and promoted long-term immune activation.

Related technique Extracellular ATP Assay, Apoptosis Plate Assay

Related Techniques (click to open/close)

Target	Kit & Probes
Extracellular ATP measurement	Extracellular ATP Assay Kit-Luminescence
Intracellular ATP measurement	ATP Assay Kit-Luminescence
Apoptosis detection in multiple samples	Annexin V Apoptosis Plate Assay Kit
Cytotoxicity measurement	Cytotoxicity LDH Assay Kit-WST
First-time autophagy research	Autophagic Flux Assay Kit
Mitochondrial Staining	MitoBright LT Green / Red / Deep Red
Mitochondrial membrane potential detection	JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
Cell proliferation	Cell Counting Kit-8

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

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）