Anoikis Assay Kit ab211153 provides a colorimetric and fluorometric format to measure anchorage-independent growth and monitor anoikis propelled cell death.
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Key facts
- Sample types
- Adherent cells
- Reactive species
- Human
What's included?
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Anoikis Assay Kit ab211153 provides a colorimetric and fluorometric format to measure anchorage-independent growth and monitor anoikis propelled cell death.
Key facts
- Sample types
- Adherent cells
- Reactive species
- Human
- Assay Platform
- Microplate reader, Fluorescence microscope
Storage
- Shipped at conditions
- Blue Ice
- Appropriate short-term storage conditions
- Multi
- Appropriate long-term storage conditions
- Multi
- Storage information
- Please refer to protocols
Notes
Anoikis Assay Kit ab211153 provides a colorimetric and fluorometric format to measure anchorage-independent growth and monitor anoikis propelled cell death.
In the anoikis assay protocol, live cells are detected with MTT or Calcein AM. Cell death is detected with Ethidium homodimer.
Background fluorescence levels are very low because the dyes are virtually non-fluorescent before interacting with cells.
Ethidium homodimer is an excellent marker for measuring dead cells. Ethidium homodimer is a red fluorescent dye that can only penetrate damaged cell membranes. Ethidium homodimer fluoresces with a 40-fold enhancement upon binding ssDNA, dsDNA, RNA, oligonucleotides, and triplex DNA.
Anoikis assay protocol summary:
- treat cells according to experimental plan
- add cells to the anchorage resistant plate and to a control plate
- culture cells for 24 - 72 hrs
- for colorimetric detection of the MTT, add MTT reagent and incubate for 2-4 hrs, then add detergent solution and incubate for 2-4 hrs, transfer to a new plate and measure absorbance
- for fluorometric detection, add calcein AM and Ethidium homodimer and incubate for 30-60 min, then analyze with a fluoresence microscope or a fluorescence microplate reader
The kit provides sufficient reagents to evaluate 24 samples on a poly-Hema coated 24-well plate or 96 samples on a hydrogel coated 96-well plate.
Adhesion to the extracellular matrix (ECM) is essential for survival and propagation of many adherent cells. Apoptosis that results from the loss of cell adhesion to the ECM, or inappropriate adhesion is defined as "anoikis". Anoikis, from the Greek word for homelessness, is involved in the physiological processes of tissue renewal and cell homeostasis.
Supplementary info
- This supplementary information is collated from multiple sources and compiled automatically.
- Activity summary
Anoikis is a programmed cell death process that occurs when epithelial cells detach from the extracellular matrix inhibiting errant cellular growth. It prevents detached cells from anchoring in inappropriate locations maintaining tissue homeostasis. Anoikis mechanisms involve various cell surface receptors and signaling pathways that detect loss of cell adhesion. Alternately known as "anoikosis" or "anoikia" this process is not associated directly with a specific protein of known mass but acts primarily in epithelial and endothelial tissues influencing numerous adhesion-related proteins.
- Biological function summary
The prevention of inappropriate anchorage-dependent growth by this process is significant in maintaining tissue integrity. Anoikis serves as a cellular safeguard eliminating cells that could contribute to tumor formation. This cell death process involves a complex interaction between integrins caspases and Bcl-2 family proteins. The interactions ensure that non-adherent cells cannot survive away from their native extracellular matrix therefore playing an essential role in development and immune-cell trafficking.
- Pathways
Anoikis is integrated within the intrinsic apoptosis pathway and the PI3K/AKT signaling pathway. These pathways regulate cell survival and death decisions based on cellular context and external signals. In the apoptosis pathway Bcl-2 family proteins mediate mitochondrial changes while the PI3K/AKT pathway influences cell survival signals that anoikis counters. Interaction between integrins and extracellular matrix components supplies survival signals whose absence triggers anoikis.
- Associated diseases and disorders
Anoikis resistance is a critical factor in cancer and fibrotic diseases. Cancer cells often acquire the ability to resist anoikis supporting metastasis by enabling survival and proliferation without matrix attachment. Proteins such as E-cadherin and focal adhesion kinase (FAK) are implicated in anoikis resistance during tumor progression. In fibrosis disrupted anoikis contributes to pathological tissue remodeling often involving overactive TGF-beta signaling pathways that include connective tissue growth factor.
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3 product images
Functional Studies - Anoikis Assay Kit (ab211153)
Anoikis assay performed on the poly-Hema coated (24-well) plate. Human foreskin fibroblasts BJ-TERT cells were seeded at 40,000 cells/well in a tissue culture control plate (top panel) or an anchorage resistant plate (poly-Hema coated) (bottom panel) and incubated for 24hours. Cells cultured in the anchorage resistant plate show signs of anoikis-like cell death (diminished calcein AM staining and increase EthD-1 staining.
Functional Studies - Anoikis Assay Kit (ab211153)
Cell viability determined by MTT in human foreskin fibroblasts BJ-TERT cells seeded at 40,000 cells/well in a 24-wp tissue culture control plate (control) or an anchorage resistant plate (poly-Hema) and incubated for 24 hours.
Functional Studies - Anoikis Assay Kit (ab211153)
Cell viability determined by MTT in human foreskin fibroblasts BJ-TERT cells seeded at 10,000 cells/well in a 96-wp tissue culture control plate (control) or an anchorage resistant plate (hydrogel) and incubated for 24hours.
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