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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.

凋亡

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.

凋亡调节:相互作用通路

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


凋亡调节:Interactive Pathway >>

如何检测凋亡

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 检测 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: 使用 Annexin V-FITC Early Apoptosis Detection Kit 对未经处理(左图)或已经喜树碱(10uM,4 小时;右图)处理的 Jurkat 细胞进行流式细胞分析。

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).

染色质凝聚 Detects apoptotic cells with condensed chromatin after staining with nuclear dyes, such as DAPI or Hoechst 33342
Hoechst 33342 #4082

Hoechst 33342 #4082: 使用 α-Tubulin (DM1A) Mouse mAb #3873(红色)和 Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb #3377(绿色)对 HeLa 细胞进行免疫荧光分析。蓝色伪彩 = Hoechst 33342(DNA 荧光染料)。

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: 使用 Cytochrome c (6H2.B4) Mouse mAb(绿色),对未经处理(左)或 Staurosporine (1 μM) #9953 和 Z-VAD (50 μM) 处理 3 小时(右)的 HeLa 细胞进行共聚焦免疫荧光分析。蓝色伪彩 = DRAQ5® #4084(DNA 荧光染料)。

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 细胞(3x105 个细胞/ml)用不同浓度的 CCCP 处理 15 分钟后再用 200 nM TMRE 标记。

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). 蓝色伪彩 = DRAQ5® #4084(DNA 荧光染料)。

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: 对未经处理 (-) 或已经以下联合处理方法处理(如图)的 HT-29 细胞进行蛋白质印迹分析:Z-VAD(20 μM,先于其他混合物 30 分钟添加;+)、人 TNF-α(hTNF-α,20 ng/ml,7 小时;+)、SM-164(100 nM,7 小时;+)和 necrostatin-1(Nec-1,50 μM,7 小时;+),使用 Phospho-RIP (Ser166) (D1L3S) Rabbit mAb(上图)或 β-Actin (D6A8) Rabbit mAb #8457(下图)。

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

MLKL (D2I6N) Rabbit mAb #14993: 使用 MLKL (D2I6N) Rabbit mAb 对不同细胞系提取物进行蛋白质印迹分析。KARPAS 细胞系来源:剑桥大学 Abraham Karpas 博士。

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). 红色 = Propidium Iodide (PI)/RNase Staining Solution #4087(DNA 荧光染料)。

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 炎性体信号转导相互作用通路
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
炎性体信号转导相互作用通路

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: 使用 Cleaved Caspase-1 (Asp296) (E2G2I) 兔单克隆抗体(上)或 Caspase-1 (E2Z1C) 兔单克隆抗体(下), 对未经处理 (-) 或已经 Lipopolysaccharides (LPS) #14011(50 ng/ml,4 小时)处理,然后使用 Nigericin处理(15 μM,45 分钟)(+) 的小鼠骨髓源性巨噬细胞 (mBMDM) 的细胞或培养基提取物进行蛋白质印迹分析。

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: 使用 Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb 对石蜡包埋的人乳腺导管癌细胞进行免疫组织化学分析。

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: 使用 LDHA (C4B5) Rabbit mAb(绿色)对 MCF-7 细胞进行共聚焦免疫荧光分析。肌动蛋白纤丝用 DY-554 Phalloidin 进行标记(红色)。蓝色伪彩 = DRAQ5® #4084(DNA 荧光染料)。

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

IL-1β (D3U3E) Rabbit mAb #12703: 使用 IL-1β (D3U3E) Rabbit mAb(绿色)对未经处理的(左)或经 LPS 处理(500 ng/ml,2 小时;右)的 THP-1 细胞进行共聚焦免疫荧光分析。使用 DY-554 phalloidin(红色)标记肌动蛋白纤丝。

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