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Tools for Studying Cell Death

Posted By Arun Kumar
Tags: Apoptosis, Autophagy, Cell Death

Overview of Cell Death

In adult tissues, cell death balances cell division. The number of cells in multicellular organisms is tightly regulated. Cellular homeostasis is achieved by controlling the rate of cell division and the rate of cell death. Cells die as a result of chemical and physical events, or “insults,” such as radiation, heat, toxic substances, bodily trauma, or injury and lack of oxygen. Injured cells swell up, burst, and spill their contents, undergoing unplanned cell death. For many years, biologists have known that cells die at predictable points during development and this represents programmed cell death (PCD).

Cell death can be classified into a number of types including apoptosis, autophagy, necrosis, and others. Of the three major pathways, Apoptosis (Programmed Cell Death Type 1) is an ordered and orchestrated cellular process that occurs in physiological and pathological conditions. It is also one of the most studied topics among cell biologists and recognized as a critical regulator of development, differentiation, regulation and function of the immune system, as well as organ and tissue proliferation/homeostasis. Dysregulation of apoptosis can play a primary or secondary role in causing diseases such as cancer. Excessive apoptosis can contribute to neurodegeneration, autoimmunity, etc. Various means of detecting apoptotic cells have been explored and made available over recent years. Autophagy, in essence, is a biological recycling mechanism where misfolded proteins are ubiquitinated and targeted for degradation by the lysosomal pathway. Autophagy (Programmed Cell Death Type 2) is selective degradation of intracellular targets, such as misfolded proteins and damaged organelles, thereby serving as an important homeostatic function. Necrosis (Programmed Cell Death Type 3) is characterized by cell swelling and destruction of the plasma membrane and subcellular organelles. Necrotic cell death is considered a heterogeneous phenomenon including both programmed and accidental cell death.


Overview of Apoptosis

Apoptotic cell death is known to involve a cascade of proteolytic events accomplished mainly by a family of cysteine proteases called caspases. These proteases are synthesized as inactive proenzymes that are activated after cleavage at specific aspartate residues. Because caspases exist in healthy cells as inactive precursors (procaspases), events that regulate caspase activation and downstream activity constitute critical control points in apoptosis. Caspases are organized in hierarchical activation networks, with some caspases playing a role as upstream initiators of caspase activation and others playing a role as downstream effectors of substrate proteolysis. There are three pathways by which caspases can be activated. The two commonly described initiation pathways are the intrinsic (or mitochondrial) and extrinsic (or death receptor) pathways of apoptosis. Both pathways eventually lead to a common pathway or the execution phase of apoptosis. A third lesser-known initiation pathway is the intrinsic endoplasmic reticulum pathway.


Methods of Detecting Apoptosis/Necrosis

The display of phosphatidylserine (PS) on the extracellular face of the plasma membrane is the hallmark of early apoptosis. Phospholipid binding proteins such as Annexin V possess a high affinity for phosphatidylserine and readily bind in the presence of Ca2+. Given that Annexin V is not cell permeable, the binding of externalized PS is selective for early apoptotic cells. As a cell trends towards necrosis, membranes continue to degrade, allowing non-permeable detection reagents, such as Necrosis Detection Reagent, to intercalate into guanine rich regions of the DNA and provide additional information. Cells that are in late apoptosis or early necrosis demonstrate signal for both Annexin V and Necrosis stain. Determining the stage of apoptosis or necrosis is of particular interest to several research areas ranging from target identification and validation to small molecule efficacy and toxicity. The binding of fluorochrome-conjugated Annexin V to exposed PS can be detected by flow cytometry or fluorescence microscopy. While fluorescence microscopy allows the visualization of the event, flow cytometry is the most useful method as it allows for a quick and accurate quantification of cells with exposed PS. Our GFP-CERTIFIED® Apoptosis/Necrosis detection kit distinguishes between healthy, early apoptotic, late apoptotic and necrotic cells and is compatible with GFP and other green fluorescent probes. We offer a range of Annexin products (Table 1).

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Table 1

Annexin V (human), (recombinant) (FITC conjugate) 

Produced in E.coli. , ≥90%, Flow Cytometry | Print as PDF
 
ALX-209-256-T100 100 tests 204.00 USD
 
ALX-209-256-3100 3x100 tests 465.00 USD
Do you need bulk/larger quantities?
 

Annexin V (human), (recombinant) (APC conjugate) 

Produced in E. coli., ≥89% (SDS-PAGE, HPLC), Flow Cytometry | Print as PDF
 
ALX-209-252-T020 20 tests 122.00 USD
 
ALX-209-252-T100 100 tests 354.00 USD
Do you need bulk/larger quantities?
 

Annexin V (human), (recombinant) (R-PE conjugate) 

Produced in E. coli., ≥89% (SDS-PAGE, HPLC), Flow Cytometry | Print as PDF
 
ALX-209-251-T020 20 tests 122.00 USD
 
ALX-209-251-T100 100 tests 354.00 USD
Do you need bulk/larger quantities?
 

Annexin V (mouse), (recombinant) (Alexa Fluor® 488 conjugate) 

Produced in E. coli. , Flow Cytometry | Print as PDF
 
ALX-209-257-T100 100 tests 310.00 USD
Do you need bulk/larger quantities?
 

Annexin V (human), (recombinant) (GST-tag) 

Produced in E. coli. Recombinant human annexin V is fused to a GST-tag., ≥95% (SDS-PAGE) | Print as PDF
 
ALX-201-116-C100 100 µg 233.00 USD
Do you need bulk/larger quantities?
 

Annexin V-Cy3 apoptosis detection kit 

Flow Cytometry, Fluorescence microscopy | Print as PDF
 
ALX-850-252-KI01 25 tests 175.00 USD
 
ALX-850-252-KI02 100 tests 319.00 USD
Do you need bulk/larger quantities?
 

Annexin V-Cy5 apoptosis detection kit 

Flow Cytometry, Fluorescence microscopy | Print as PDF
 
ALX-850-254-KI01 25 tests 174.00 USD
 
ALX-850-254-KI02 100 tests 354.00 USD
Do you need bulk/larger quantities?
 

Annexin V-EGFP apoptosis detection kit 

Flow Cytometry, Fluorescence microscopy | Print as PDF
 
ALX-850-253-KI02 100 tests 354.00 USD
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Annexin V-FITC apoptosis detection kit plus 

Flow Cytometry, Fluorescence microscopy | Print as PDF
 
ALX-850-255-KI02 100 tests 336.00 USD
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Annexin V (human) ELISA kit 

ELISA | Print as PDF
 
ALX-850-049-KI01 96 wells 703.00 USD
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Genomic DNA Fragmentation and Comet SCGE Assay Kit

Assays to evaluate the integrity of genomic DNA, or to assess the presence of oxidized DNA, are frequently used as a means of verifying the onset of apoptosis or DNA damage. The COMET SCGE Assay measures DNA damage by fluorescently detecting the integrity of DNA liberated from cells embedded in low melting point agarose. Upon electrophoresis, fragmented DNA produces a characteristic "comet" shaped tail as small DNA fragments migrate in the gel more rapidly than intact genomic DNA. It is possible to differentiate various forms of DNA strand breakage dependent on the pH of electrophoresis. Under alkaline conditions (pH >13) information regarding single-strand breakage, double-strand breakage, excision repair sites, and alkaline-labile sites can be obtained. Under neutral conditions, only double-strand DNA breakage is observed and is therefore considered a method for detection of apoptosis. This method is useful in assessing viability of cells and in differentiating types of cell death - apoptosis versus necrosis. The COMET SCGE Assay is a fast and simple electrophoresis method to detect and quantitate DNA fragmentation in cells associated with DNA damage and apoptosis. Our unique CYGREEN® Nucleic acid dye provides improved sensitivity for DNA visualization compared to ethidium bromide.


Nuclear Condensation

Characteristic apoptotic features include cell membrane blebbing, cell shrinkage, chromatin condensation and DNA fragmentation and packaging into apoptotic bodies. Three stages of apoptotic chromatin condensation have been defined based upon morphological and biochemical criteria: stage 1 “ring” condensation, stage 2 “necklace” condensation, and stage 3 “nuclear collapse/disassembly”. Our NUCLEAR-ID® Green Chromatin Condensation Detection Kit provides a convenient approach for analysis of late stage apoptosis by flow cytometry and microscopy. The DNA intercalating dye brightly stains the compacted chromatin of apoptotic cells compared to the healthy cells. The kit is suitable for differentiating between healthy and apoptotic cells with condensed nuclei.


Mitochondrial Perturbations and Activation of Intrinsic Pathway Activation

The loss of the mitochondrial membrane potential (MMP) is often associated with the early stages of apoptosis. The collapse of MMP coincides with the opening of the mitochondrial permeability transition pores, leading to the release of cytochrome C into the cytosol, which in turn triggers other downstream events in the apoptotic cascade. The MITO-ID® membrane potential detection kit contains a cationic carbocyanine dye that fluoresces either green or orange, depending upon MMP status. This is the only assay that monitors energetic status using an easy-to-use protocol. In energized cells, the MITO-ID® Membrane Potential dye exists as a green-fluorescent monomer in the cytosol and accumulates as orange fluorescent aggregates in the mitochondria. However, in apoptotic and necrotic cells, this dye exits mitochondria and exists primarily as green-fluorescent monomers in the cytosol. A cell-impermeant DNA intercalator, the necrosis detection reagent, similar to the red-emitting dye propidium iodide, is used to monitor late-stage apoptosis and necrotic cell death. We offer a range of high throughput assays for mitochondrial function (Table 2).

Brochures
Monitor programmed cell death from membrane to nucleus
 

Table 2

MITO-ID® Green detection kit  

Photostable,  non-toxic and selective mitochondrial dye that stains regardless of membrane potential
Fluorescence microscopy, Fluorescent detection | Print as PDF
 
ENZ-51022-0100 100 tests 87.00 USD
 
ENZ-51022-K500 500 tests 241.00 USD
Do you need bulk/larger quantities?
 

MITO-ID® Red detection kit (GFP-CERTIFIED®) 

Photostable,  non-toxic and selective mitochondrial dye that stains regardless of membrane  potential
Fluorescence microscopy, Fluorescent detection | Print as PDF
 
ENZ-51007-0100 100 tests 83.00 USD
 
ENZ-51007-500 500 tests 242.00 USD
Do you need bulk/larger quantities?
 

MITO-ID® Extracellular O2 Sensor Kit (High Sensitivity) 

High-throughput cell-based assay used to detect mitochondria dysfunction through real-time oxygen consumption measurements in whole cells
Fluorescent detection, HTS, Cell-based assays | Print as PDF
 
ENZ-51045-K500 500 Reactions Inquire for pricing
 
ENZ-51045-K100 100 Reactions Inquire for pricing
Do you need bulk/larger quantities?
 

MITO-ID® Extracellular pH Sensor Kit 

A pH sensitive phosphorescent probe that can monitor cellular lactic acid extrusion, resulting from glycolysis, as a measure of glycolytic flux  (ECA/ECAR)
Fluorescent detection, HTS | Print as PDF
 
ENZ-51048 1 Kit Inquire for pricing
Do you need bulk/larger quantities?
 

Apoptosis-related Proteases: Caspases

Caspases serve as signaling mediators that orchestrate apoptotic execution pathways by cleaving a subset of cellular proteins. More than 100 substrates have been identified thus far. Mammalian caspases can be subdivided into three functional groups: initiator caspases (Caspase 2, 8, 9 and 10), executioner caspases (Caspase 3, 6 and 7), and inflammatory caspases (Caspase 1, 4, 5, 11 and 12). Initiator caspases initiate the apoptosis signal while the executioner caspases carry out the mass proteolysis that leads to apoptosis. Inflammatory caspases do not function in apoptosis but are instead involved in inflammatory cytokine signaling. Activation of caspases can be detected using multiple methods depending on the instrumentation available and how the sample has been prepared. Highlighted below are a subset of the caspase activity kits and active enzymes we offer (Table 3).

Table 3

Caspase-2 assay kit for drug discovery 

Colorimetric detection, Fluorescent detection, HTS, Activity assay | Print as PDF
 
BML-AK702-0001 96 wells 543.00 USD
Do you need bulk/larger quantities?
 

Caspase-3 cell assay kit 

Colorimetric detection, Fluorescent detection, HTS, Activity assay | Print as PDF
 
BML-AK703-0001 96 wells 412.00 USD
Do you need bulk/larger quantities?
 

Caspase-3 assay kit for drug discovery 

Colorimetric detection, Fluorescent detection, HTS, Activity assay | Print as PDF
 
BML-AK700-0001 96 wells 543.00 USD
Do you need bulk/larger quantities?
 

Caspase-8 assay kit for drug discovery 

Colorimetric detection, Fluorescence microscopy, HTS | Print as PDF
 
BML-AK715-0001 96 wells 543.00 USD
Do you need bulk/larger quantities?
 

Caspase-1 (human), (recombinant) 

Highly active caspase essential for apoptosis.
Produced in E. coli. cDNA encodes residues identical to Asn120-His404 (C-terminus) at Genbank Accession No. M87507, except for an Asp381 to Glu change, introduced to stabilize the enzyme against autoproteolysis., ≥90% (SDS-PAGE) | Print as PDF
 
BML-SE168-5000 5000 U 412.00 USD
Do you need bulk/larger quantities?
 

Caspase-3 (human), (recombinant) 

Produced in E. coli. Caspase-3 (CPP32; Yama; apopain) from human cDNA. The enzyme was cleaved and activated from the proenzyme, ≥95% (SDS-PAGE) | Print as PDF
 
BML-SE169-5000 5000 U 412.00 USD
Do you need bulk/larger quantities?
 

Caspase-4 (human), (recombinant) 

Produced in E. coli., ≥90% (SDS-PAGE) | Print as PDF
 
BML-SE176-5000 5000 U 412.00 USD
Do you need bulk/larger quantities?
 

Caspase-5 (human), (recombinant) 

Produced in E. coli, ≥80% (SDS-PAGE) | Print as PDF
 
BML-SE171-5000 5000 U 412.00 USD
Do you need bulk/larger quantities?
 

Caspase-6 (human), (recombinant) 

Highly, active caspase-6 for the study of enzyme kinetics or screening of inhibitors
Produced in E. coli., ≥80% (SDS-PAGE) | Print as PDF
 
BML-SE170-5000 5000 U 409.00 USD
Do you need bulk/larger quantities?
 

Caspase-8 (human), (recombinant) 

Highly active caspase-8 for the study of enzyme regulation and kinetics
Produced in E. coli, ≥90% (SDS-PAGE) | Print as PDF
 
BML-SE172-5000 5000 U 412.00 USD
Do you need bulk/larger quantities?
 

The use of activators or inhibitors for specific steps of the apoptosis cascade is essential in order to provide positive and negative controls. These ensure that the assay works, and confirm that apoptosis is occurring. We offer ready-to-use caspase inhibitors with high purity for modulation of the caspase signaling cascade (Table 4).

Autophagy Detection

Autophagy, also known as caspase-independent form of cell death or Type 2 PCD, is a physiologically and evolutionarily conserved process maintaining cellular homeostasis and genomic integrity by engulfing and degrading malfunctioning organelles and proteins in living cells. Under physiological conditions, autophagy plays a variety of important roles including maintenance of the amino acid pool during starvation, damaged protein and organelle turnover, prevention of neurodegeneration, tumor suppression, cellular differentiation, and clearance of intracellular microbes and regulation of innate and adaptive immunity. Various cytoplasmic constituents, including organelles and long-lived proteins, are sequestered into double-membraned autophagosomes, which subsequently fuse with lysosomes where their contents are degraded. Enzo Life Sciences’ CYTO-ID® Autophagy detection kit 2.0 is a no transfection assay that contains a photostable dye that specifically stains autophagic vesicles. The detection kit has been optimized for detection of autophagy in live cells by fluorescence microscopy, flow cytometry, and fluorescence microplate assay. The assay provides a rapid, specific and quantitative approach for monitoring autophagic activity at the cellular level.

Brochures
Autophagy
 

Table 4

Ac-DEVD-CHO 

Caspase-3 inhibitor
169332-60-9, ≥95% (HPLC) | Print as PDF
 
ALX-260-030-M001 1 mg 63.00 USD
 
ALX-260-030-M005 5 mg 231.00 USD
Do you need bulk/larger quantities?
 

Ac-YVAD-CMK 

Inhibitor of caspase-1 and -4
178603-78-6, ≥96% (HPLC) | Print as PDF
 
ALX-260-028-M001 1 mg 89.00 USD
 
ALX-260-028-M005 5 mg 326.00 USD
Do you need bulk/larger quantities?
 

Z-Asp-2,6-dichlorobenzoyloxymethylketone 

Caspase inhibitor
153088-73-4, ≥98% (TLC) | Print as PDF
 
ALX-260-029-M010 10 mg 131.00 USD
 
ALX-260-029-M050 50 mg 457.00 USD
Do you need bulk/larger quantities?
 

Z-DEVD-FMK (Ready-to-Use) 

Inhibitor of caspase-3 and -7
≥98% (HPLC) | Print as PDF
 
ALX-260-141-R100 100 µl 2mM 189.00 USD
 
ALX-260-141-R020 20 µl 10mM 189.00 USD
Do you need bulk/larger quantities?
 

Z-VAD-FMK (Ready-to-Use) 

Caspase inhibitor
Synthetic, ≥99% (TLC) | Print as PDF
 
ALX-260-138-R100 100 µl 2mM 189.00 USD
 
ALX-260-138-R020 20 µl 10mM 189.00 USD
Do you need bulk/larger quantities?
 

Enzo Life Sciences offers a range of products for your Cellular Analysis research needs. Enzo’s solutions for cellular analysis enable investigation of basic cell biology, its relation to disease pathology, and the development of novel small molecule, biotherapeutic, and cell-based therapies. Built upon our diverse scientific expertise and technology platforms, we support cellular analysis on the genomic, protein, small molecule and whole cell levels. As Scientists Enabling Scientists, we realize the value in providing relevant information to our customers working in the fields of life sciences, drug development and clinical research. We are happy to provide simple yet useful tips and guidance for your research needs. Please check out our successful research tips or contact our Technical Support Team for further assistance.

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