APOPTOSIS
Apoptosis는 다세포 생물체에서 볼 수 있는 Programmed cell death의 일종으로 ATP-dependent하며 치밀한 유전적 프로그램을 따르는 자발적 세포제거 과정입니다. 이 과정은 세포질 수축, DNA fragmentation, Nuclear condensation, 세포막의 변화, 세포사멸체 형성, 그리고 최종적으로 Macrophages phagocytose가 일어나면서 종료됩니다.
- Activation of apoptosis signaling cascades
- Pannexin channel “find me” signal release
- Phosphatidylserine (PS) exposure
- Mitochondria outer membrane permeability
- Release of cytochrome c from mitochondria
- Activation of initiator and effector caspases
- Cleavage of specific caspase substrates
- Chromatin condensation and DNA fragmentation
- Membrane blebbing
- Formation of apoptotic bodies
Simultaneous Detection of Multiple Changes in Apoptosis Pathways
세포 사멸은 다양한 경로로 발생할 수 있으므로 in vitro에서 multiple apoptosis marker를 test하여 apoptosis mechanism을 확인해야 합니다. Cayman에서는 이와 관련된 제품들을 제공하고 있습니다.
Cell populations induced to undergo apoptosis by various stimuli: Untreated (NT); Ultraviolet light (UV); Etoposide (ETO); Anti-Fas CH-11
Contains
DAPI – DNA stain measures membrane permeability
Annexin V – PS exposure and loss of membrane asymmetry
TMRE – Mitochondrial membrane potential
TO-PRO®-3 – Identifies active pannexin channels
A) Untreated. B) RAW 264.7 cells stimulated to undergo apoptosis by UV concurrently decrease TMRE staining and increase Annexin V FITC staining.
Contains
Hoechst 33342 – Demonstrates nuclear morphology
Annexin V – PS exposure and loss of membrane asymmetry
TMRE – Mitochondrial membrane potential
RedDot™2 – DNA stain measures membrane permeability
Pro- and Anti-Apoptotic Signaling Pathways
The pro- and anti-apoptotic Bcl-2 family of proteins regulates commitment to cell death through controlling mitochondrial integrity. Cleavage or oligomerization as well as the abundance of these proteins can mark apoptosis.
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Cell Membrane Alterations
PS migrates to the outer plasma membrane in apoptosis, causing a loss of membrane asymmetry. Annexin V binds to exposed PS and can be paired with a membrane-impermeable dye to distinguish between intact, apoptotic, and necrotic cells.
Annexin V Assays
| Item No. | Product Name | Includes | Ex/Em Filters |
| 601410 | Annexin V APC Assay Kit | APC-conjugated annexin V and DAPI | 633/700 nm (APC); 350/450 nm (DAPI) |
| 600300 | Annexin V FITC Assay Kit | FITC-conjugated annexin V and PI | 488/525 nm (FITC); 655-730 nm (PI) |
| 601420 | Annexin V PE Assay Kit | PE-conjugated annexin V and DAPI | 488/585 nm (PE); 350/450 nm (DAPI) |
Mitochondrial Changes
Mitochondrial outer membrane permeabilization causes a decrease in transmembrane potential (Δψm) and the release of cytochrome c. Δψm is assessed using positively charged dyes, such as JC-1 and TMRE, that accumulate inside active mitochondria.
Mitochondrial Membrane Potential Assays
Caspase Activaction
Release of cytochrome c from the mitochondria promotes caspase-9 activation via Apaf-1, which then activates caspases-3 and -7. Caspase activation can be confirmed by detecting cleaved PARP1 or fluorogenic substrates.
Capase Activity Assay & Related Antibody
Chromatin Condensation & DNA Fragmentation
The condensation of chromatin is accompanied by the hydrolysis of nuclear DNA into a ladder of fragments that can be detected by gel electrophoresis. The 3’ ends of DNA fragments can also be labeled with deoxyuridine. Cell-permeable Hoechst 33342 is often used to identify nuclear condensation by microscopic analysis.
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