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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.

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

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.

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

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

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

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

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 is a type of passive 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.

Types of 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 and RIP3 kinases Protein coProtein complex containing RIP and RIP3 kinases that initiates necroptosis
RIP3 (D4G2A) Rabbit mAb #95702

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 and Phospho-RIP3 kinases Phosphorylation of RIP kinases are used as an indicator of activation of necroptotic signaling events.
Phospho-RIP (Ser166) (D1L3S) Rabbit mAb #65746

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).

Mixed lineage kinase domain-like protein MLKL Downstream protein target of RIP3
MLKL (D2I6N) Rabbit mAb #14993

MLKL (D2I6N) Rabbit mAb #14993: Western blot analysis of extracts from various cell lines using MLKL (D2I6N) Rabbit mAb. KARPAS cell Line source: Dr. Abraham Karpas at the University of Cambridge.

Phospho-MLKL Phosphorylation of MLKL is a marker for necroptotic cells
Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb #37333

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
Inflammasome Signaling Interactive Pathway >>
Caspase-1 activity Cleavage of caspase-1 occurs during pyroptosis Cleaved Caspase-1 (Asp296) (E2G2I) Rabbit mAb #89332
Gasdermin-D cleavage Cleavage of gasdermin-D occurs during pyroptosis Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb #36425
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

Inflammasome Signaling Interactive Pathway >>

Caspase-1 activity Cleavage of caspase-1 occurs during pyroptosis
Cleaved Caspase-1 (Asp296) (E2G2I) Rabbit mAb #89332

Cleaved Caspase-1 (Asp296) (E2G2I) Rabbit mAb #8933: 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-D cleavage Cleavage of gasdermin-D occurs during pyroptosis
Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb #36425

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

HMGB1 (D3E5) Rabbit mAb #6893: Immunohistochemical analysis of paraffin-embedded human lung carcinoma 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

LDHA (C4B5) Rabbit mAb #3582: Confocal immunofluorescent analysis of MCF-7 cells using LDHA (C4B5) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Interleukin-1β (IL-1β) Proinflammatory cytokine released during necrosis
IL-1β (D3U3E) Rabbit mAb #12703

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).