Overview

  • Product name

    Anoikis Assay Kit
  • Sample type

    Adherent cells
  • Species reactivity

    Reacts with: Human
  • Product overview

    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

  • Notes

    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.

  • Platform

    Microplate reader, Fluorescence microscope

Properties

  • Storage instructions

    Please refer to protocols.
  • Components 24 tests 96 tests
    500X Calcein AM 1 x 50µl 1 x 50µl
    500X Ethidium Homodimer 1 x 50µl 1 x 50µl
    Anchorage Resistant Plate 1 unit 1 unit
    Detergent Solution 1 x 25ml 1 x 25ml
    MTT Solution 3 x 1ml 3 x 1ml

Images

  • 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.

  • 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.

  • 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.

Protocols

References

ab211153 has not yet been referenced specifically in any publications.

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