Overview of Cell Death

Mechanisms and Types of Cell Death

Cells can die in a number of different manners, depending on the cellular context and triggering stimulus.

Types of Cell Death

Cell death mechanisms include:

Apoptosis - programmed cell death that occurs during growth and development and can also occur in response to harmful environmental stimuli.
Necrosis - can be a passive or an active, regulated process such as necroptosis or pyroptosis.

Different assays can be used to determine the mechanism of cell death or rule out a mechanism of cell death within a cellular population.

Apoptosis

Apoptosis is a highly regulated form of programmed cell death that occurs in multicellular organisms during development, throughout the lifespan, and in response to cellular stress. Apoptosis is mediated by a family of proteolytic enzymes called caspases. Other proteins, including proapoptotic and antiapoptotic proteins, also play important roles.

Dysregulation of apoptosis occurs in several disease states, including autoimmune disorders, neurodegenerative diseases, and cancer.

Apoptosis is a form of programmed cell death mediated by caspases and a host of other proteins.

Regulation of Apoptosis: Interactive Pathway >>

Apoptosis is a form of programmed cell death mediated by caspases and a host of other proteins.

Regulation of Apoptosis: Interactive Pathway >>

How to Measure Apoptosis

Apoptotic cells can be distinguished from viable cells by phenotypic changes and activity of certain proteins. Several different methods can be used to analyze and measure levels of apoptosis within a population. These assays include:

Assay	What is Measured
Annexin V assay	Detects changes that occur to the lipid bilayer early in apoptosis	Annexin V-FITC Early Apoptosis Detection Kit #6592: Flow cytometric analysis of Jurkat cells untreated (left) or treated with camptothecin (10uM, 4 hr; right) using Annexin V-FITC Early Apoptosis Detection Kit. Confocal immunofluorescent analysis of normal rat cerebellum, hippocampus and striatum using α-Synuclein (D37A6) XP^® Rabbit mAb #4179 (green). Blue pseudocolor = DRAQ5^® #4084 (fluorescent DNA dye).
TUNEL Kits	DNA fragmentation which is a hallmark of apoptosis	DNA fragmentation which is a hallmark of apoptosis
Caspase cleavage	Cleavage of caspase-3, caspase activity assay, and cleavage of other caspases and PARP are frequently used readouts for apoptosis	Cleaved Caspase-3 (Asp175) Antibody - #9661: Western blot analysis of extracts from HeLa, NIH/3T3 and C6 cells untreated, staurosporine-treated (3hrs, 1 μM in vivo) or cytochrome c-treated (1hr, 0.25 mg/ml in vitro), using Caspase-3 Antibody #9662 (upper) or Cleaved Caspase-3 (Asp175) Antibody (lower).
Chromatin condensation	Detects apoptotic cells with condensed chromatin after staining with nuclear dyes, such as DAPI or Hoechst 33342	Hoechst 33342 #4082: Immunofluorescent analysis of HeLa cells using α-Tubulin (DM1A) Mouse mAb #3873 (red) and Phospho-Histone H3 (Ser10) (D2C8) XP^® Rabbit mAb #3377 (green). Blue pseudocolor = Hoechst 33342 (fluorescent DNA dye).
Cytochrome c release	Translocation of cytochrome c from mitochondria to the cytoplasm is a hallmark feature of apoptotic cells	Cytochrome c (6H2.B4) Mouse mAb #129638: Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Staurosporine (1 μM) #9953 and Z-VAD (50 μM) for 3 hours (right), using Cytochrome c (6H2.B4) Mouse mAb (green). Blue pseudocolor= DRAQ5^® #4084 (fluorescent DNA dye).
Mitochondrial membrane potential assay	Depolarization and subsequent decrease of the mitochondrial membrane potential is a hallmark feature of apoptotic cells	Mitochondrial Membrane Potential Assay Kit (II) #13296: HeLa cells (3x10⁵ cell/ml) were treated with various concentrations of CCCP for 15 minutes prior to labeling with 200 nM TMRE.

Assay	What is Measured
Annexin V assay	Detects changes that occur to the lipid bilayer early in apoptosis
Annexin V-FITC Early Apoptosis Detection Kit #6592: Flow cytometric analysis of Jurkat cells untreated (left) or treated with camptothecin (10uM, 4 hr; right) using Annexin V-FITC Early Apoptosis Detection Kit.
TUNEL Kits	DNA fragmentation which is a hallmark of apoptosis
DNA fragmentation which is a hallmark of apoptosis
Caspase cleavage	Cleavage of caspase-3, caspase activity assay, and cleavage of other caspases and PARP are frequently used readouts for apoptosis
Cleaved Caspase-3 (Asp175) Antibody - #9661: Western blot analysis of extracts from HeLa, NIH/3T3 and C6 cells untreated, staurosporine-treated (3hrs, 1 μM in vivo) or cytochrome c-treated (1hr, 0.25 mg/ml in vitro), using Caspase-3 Antibody #9662 (upper) or Cleaved Caspase-3 (Asp175) Antibody (lower).
Chromatin condensation	Detects apoptotic cells with condensed chromatin after staining with nuclear dyes, such as DAPI or Hoechst 33342
Hoechst 33342 #4082: Immunofluorescent analysis of HeLa cells using α-Tubulin (DM1A) Mouse mAb #3873 (red) and Phospho-Histone H3 (Ser10) (D2C8) XP^® Rabbit mAb #3377 (green). Blue pseudocolor = Hoechst 33342 (fluorescent DNA dye).
Cytochrome c release	Translocation of cytochrome c from mitochondria to the cytoplasm is a hallmark feature of apoptotic cells
Cytochrome c (6H2.B4) Mouse mAb #129638: Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Staurosporine (1 μM) #9953 and Z-VAD (50 μM) for 3 hours (right), using Cytochrome c (6H2.B4) Mouse mAb (green). Blue pseudocolor= DRAQ5^® #4084 (fluorescent DNA dye).
Mitochondrial membrane potential assay	Depolarization and subsequent decrease of the mitochondrial membrane potential is a hallmark feature of apoptotic cells
Mitochondrial Membrane Potential Assay Kit (II) #13296: HeLa cells (3x10⁵ cell/ml) were treated with various concentrations of CCCP for 15 minutes prior to labeling with 200 nM TMRE.

Necrosis

Necrosis has been classically defined as an unprogrammed cell death that occurs following acute injury or infection or when apoptosis is inhibited and is characterized by cellular swelling and lysis. Necrotic cells release intracellular contents into the surrounding environment, which activates an inflammatory response to recruit phagocytes to clear dead cells. Uncontrolled, however, necrosis can cause severe tissue damage, such as gangrene.

Necrotic Cell Death Signaling Interactive Pathway >>

While it was previously thought that necrosis was passive and unprogrammed, recent data have uncovered different types of regulated necroptotic pathways.

Types of Regulated Necrosis

In addition to necrosis, other lytic cell death mechanisms include:

Necroptosis - a programmed and regulated form of necrosis which requires RIP3 and MLKL and is activated by pro-inflammatory signaling as well as ischemic injury and viral infection.
Pyroptosis - a form of programmed lytic cell death that typically occurs in immune cells in response to microbial or viral infection and requires caspase-1 and gasdermin-D

How to Measure Necroptosis:

Necroptosis Marker	Necroptosis Marker Description
RIP	Ser/Thr kinase that regulates inflammation and cell death	RIP (D94C12) XP Rabbit mAb #3493: Confocal immunofluorescent analysis of OVCAR8 cells using RIP (D94C12) XP^® Rabbit mAb (green). Blue pseudocolor = DRAQ5^® #4084 (fluorescent DNA dye).
RIP3	Ser/Thr kinase that is required for necroptosis	RIP3 (D4G2A) Rabbit mAb #95702: Confocal immunofluorescent analysis of L-929 using RIP3 (D4G2A) Rabbit mAb (green). Blue pseudocolor = DRAQ5^® #4084 (fluorescent DNA dye).
Phospho-RIP	Activated RIP associates with RIP3 to trigger necroptosis	Phospho-RIP (Ser166) (D1L3S) Rabbit mAb #65746: Western blot analysis of HT-29 cells, untreated (-) or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), human TNF-α (hTNF-α, 20 ng/ml, 7 hr; +), SM-164 (100 nM, 7 hr; +), and necrostatin-1 (Nec-1, 50 μM, 7 hr; +), using Phospho-RIP (Ser166) (D1L3S) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Phospho-RIP3	Activation of RIP3 leads to phosphorylation of MLKL	Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb #93654: Western blot analysis of HT-29 cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), and SM-164 (100 nM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb. To confirm phospho-specificity, membranes were either untreated (left) or treated with Calf Intestinal Phosphatase (CIP; right).
MLKL	Downstream protein target of RIP3	Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc-tagged full-length human MLKL protein (hMLKL-Myc; +), using MLKL (E7V4W) Mouse mAb (upper) or GAPDH (D16H11) XP^® Rabbit mAb #5174 (lower).
Phospho-MLKL	Phosphorylation of MLKL leads to pore formation and is a marker for necroptotic cells	Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb #37333: Confocal immunofluorescent analysis of L-929 cells, pre-treated with Z-VAD (20 μM, 30 min) followed by treatment with SM-164 (100 nM) and Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/mL, 2.5 hr;) and then post-processed using Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb (green). Red = Propidium Iodide (PI)/RNase Staining Solution #4087 (fluorescent DNA dye).

Necroptosis Marker	Necroptosis Marker Description
RIP	Ser/Thr kinase that regulates inflammation and cell death
RIP (D94C12) XP^® Rabbit mAb #3493: Confocal immunofluorescent analysis of OVCAR8 cells using RIP (D94C12) XP^® Rabbit mAb (green). Blue pseudocolor = DRAQ5^® #4084 (fluorescent DNA dye).
RIP3	Ser/Thr kinase that is required for necroptosis
RIP3 (D4G2A) Rabbit mAb #95702: Confocal immunofluorescent analysis of L-929 using RIP3 (D4G2A) Rabbit mAb (green). Blue pseudocolor = DRAQ5^® #4084 (fluorescent DNA dye).
Phospho-RIP	Activated RIP associates with RIP3 to trigger necroptosis
Phospho-RIP (Ser166) (D1L3S) Rabbit mAb #65746: Western blot analysis of HT-29 cells, untreated (-) or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), human TNF-α (hTNF-α, 20 ng/ml, 7 hr; +), SM-164 (100 nM, 7 hr; +), and necrostatin-1 (Nec-1, 50 μM, 7 hr; +), using Phospho-RIP (Ser166) (D1L3S) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Phospho-RIP3	Activation of RIP3 leads to phosphorylation of MLKL
Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb #93654: Western blot analysis of HT-29 cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), and SM-164 (100 nM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb. To confirm phospho-specificity, membranes were either untreated (left) or treated with Calf Intestinal Phosphatase (CIP; right).
MLKL	Downstream protein target of RIP3
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc-tagged full-length human MLKL protein (hMLKL-Myc; +), using MLKL (E7V4W) Mouse mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Phospho-MLKL	Phosphorylation of MLKL leads to pore formation and is a marker for necroptotic cells
Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb #37333: Confocal immunofluorescent analysis of L-929 cells, pre-treated with Z-VAD (20 μM, 30 min) followed by treatment with SM-164 (100 nM) and Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/mL, 2.5 hr;) and then post-processed using Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb (green). Red = Propidium Iodide (PI)/RNase Staining Solution #4087 (fluorescent DNA dye).

How to Measure Pyroptosis:

Pyroptosis Marker	Pyroptosis Marker Description
Inflammasome formation	Pyroptosis is characterized by the formation of the inflammasome; a marker for the inflammasome is NLRP3	Inflammasome Signaling Interactive Pathway >>
Caspase-1 activity	Cleavage of caspase-1 is a marker for its activity. Activated caspase-1 cleaves IL-1β and gasdermin D	Cleaved Caspase-1 (Asp296) (E2G2I) Rabbit mAb #89332: Western blot analysis of cell extracts from the cells or media from mouse bone marrow derived macrophages (mBMDM), untreated (-) or treated with Lipopolysaccharides (LPS) #14011 (50 ng/ml, 4 hr) followed by Nigericin (15 μM, 45 min) (+), using Cleaved Caspase-1 (Asp296) (E2G2I) Rabbit mAb (upper), or Caspase-1 (E2Z1C) Rabbit mAb (lower).
Gasdermin cleavage	Cleavage of gasdermin-D occurs during pyroptosis lead to pore formation	Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb #36425: Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.

Pyroptosis Marker	Pyroptosis Marker Description
Inflammasome formation	Pyroptosis is characterized by the formation of the inflammasome; a marker for the inflammasome is NLRP3
Inflammasome Signaling Interactive Pathway >>
Caspase-1 activity	Cleavage of caspase-1 is a marker for its activity. Activated caspase-1 cleaves IL-1β and gasdermin D
Cleaved Caspase-1 (Asp296) (E2G2I) Rabbit mAb #89332: Western blot analysis of cell extracts from the cells or media from mouse bone marrow derived macrophages (mBMDM), untreated (-) or treated with Lipopolysaccharides (LPS) #14011 (50 ng/ml, 4 hr) followed by Nigericin (15 μM, 45 min) (+), using Cleaved Caspase-1 (Asp296) (E2G2I) Rabbit mAb (upper), or Caspase-1 (E2Z1C) Rabbit mAb (lower).
Gasdermin cleavage	Cleavage of gasdermin-D occurs during pyroptosis lead to pore formation
Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb #36425: Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.

How to Assess Necrosis:

Necrosis Marker	Necrosis Marker Description
High mobility group protein B1 (HMGB1)	Nuclear protein that is released into the extracellular environment during necrotic, but not apoptotic, cell death	HMGB1 (D3E5) Rabbit mAb #6893: Western blot analysis of extracts from various cell lines using HMGB1 (D3E5) Rabbit mAb.
Lactate dehydrogenase A (LDHA)	Cytosolic enzyme released into the extracellular space during necrotic cell death	LDHA (C4B5) Rabbit mAb #3582: Western blot analysis of extracts from various cell types using LDHA (C4B5) Rabbit mAb.
Interleukin-1β (IL-1β)	Proinflammatory cytokine released during necrosis	IL-1β (D3U3E) Rabbit mAb #12703: Confocal immunofluorescent analysis of THP-1 cells, untreated (left) or LPS-treated (500 ng/ml, 2 hr; right), using IL-1β (D3U3E) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).

Necrosis Marker	Necrosis Marker Description
High mobility group protein B1 (HMGB1)	Nuclear protein that is released into the extracellular environment during necrotic, but not apoptotic, cell death
HMGB1 (D3E5) Rabbit mAb #6893: Western blot analysis of extracts from various cell lines using HMGB1 (D3E5) Rabbit mAb.
Lactate dehydrogenase A (LDHA)	Cytosolic enzyme released into the extracellular space during necrotic cell death
LDHA (C4B5) Rabbit mAb #3582: Western blot analysis of extracts from various cell types using LDHA (C4B5) Rabbit mAb.
Interleukin-1β (IL-1β)	Proinflammatory cytokine released during necrosis
IL-1β (D3U3E) Rabbit mAb #12703: Confocal immunofluorescent analysis of THP-1 cells, untreated (left) or LPS-treated (500 ng/ml, 2 hr; right), using IL-1β (D3U3E) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).