Apoptosis Assays

Detection of programmed cell death or apoptosis by flow cytometry.

  • Highly sensitive assays to detect early to late apoptotic stages

  • Large number of assays to detect different parameters

  • Very well validated in flow cytometry with many different type of cells

  • Validated with many different types of drugs

  • Different fluorescent options for multiple laser excitation sources

Apoptotic cell detection: Diagnosis Application

Apoptosis is a regulated process of cell death that occurs during embryonic development as well as maintenance of tissue homeostasis. The appearance of non-regulated apoptosis involves the possibility of different diseases, such as neurodegenerative diseases and cancer.

Neurological Diseases

Cancer Research

Sperm Viability

Drugs development

Understanding the mechanisms of programmed cell death or apoptosis, is an important aspect in the research of e.g., tissue homeostasis, tumour diseases, or the toxicity and efficacy of drugs, among others.

Why is annexin V Staining used to detect apoptosis?

The human vascular anticoagulant, annexin V is a 35-36KDa Ca2+ dependent phospholipids binding protein that has a high affinity for PS. In healthy cells, PS is located on the cytoplasmic surface of the plasma membrane.

However, during the earlies apoptosis, phosphatidylserine (PS), normally located on the cytoplasmic surface of the cell membrane, becomes exposed to the extracellular environment, causing changes in the membrane asymmetry and permeability.

Highly sensitive assays to detect early to late apoptotic stages

The membrane assays from Immunostep are based on the combined detection of apoptosis and necrosis by fluorescent vital double staining with Annexin V and PI or 7-AAD. Annexin V binds to phosphatidylserine which is translocated outside of the membrane during apoptosis, while PI or 7-AAD can only penetrate into necrotic cells.

Flow cytometry-based apoptosis detection

The flow cytometric analysis of apoptosis is based on different detection principles. Due to the complexity of cell death cascades the multi-parameter kits from Immunostep are particularly well suited to assess the apoptosis.

Figure 2: Mitochondrial cationic Dye process Illustration

Mitochondrial Assays: detection of early apoptotic cells.

The dysfunction of the mitochondria is a special feature of earlier stages of apoptosis. MitoStep apoptosis kits measure changes in the mitochondrial membrane potential with a solution of the cationic cyanine dye DiIC1(5).

In healthy cells, especially in mitochondria with active membrane potentials, the dye accumulates. During apoptosis the DiIC1(5) fluorescence intensity decreases and thus there is an increase of cells with lower DiIC1(5) fluorescence. 

MitoStep Apoptotic Kits:

Depolarization of the sub mitochondrial and therefore changes in the mitochondrial membrane have been describe during early stages of apoptosis. That is the reason why mitochondrial uptake of cationic dyes is a possible source of fluoresce variance.

DiIC1(5) stained cells can be visualized by excitation with a 633 nm laser and emission at 658 nm, and they be combined with FITC or PE staining.

Figure 3: Jurkat Cells T/cell Leukemia, human (treated with 6um Camptothecin for four hours/both panel.

Product References

FITC-Fluorescein isothiocyanate (Blue Laser) | More info

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PE- R-Phycoerythrin (Blue Laser) | More Info

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Dy634- Dyomics 634 (Red Laser) | More info

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CF-blue – CF405M (Violet Laser) |More info

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CF-blue – CF405M (Violet Laser) |More info

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MitoStep | DilC1(5), 500 ulof10uM in DMSO | More info

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MitoStep + FITC Apoptosis detection kit | DilC1(5), 500 ulof10uM in DMSO, ANXVKF-FITC Apoptosis Detection Kit | More info

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MitoStep + PE Apoptosis Detection Kit | DilC1(5), 500 ulof10uM in DMSO, ANXVKPE Apoptosis detection Kit | More info

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