Cold Spring Harbor Protocols Download full-text
Cold Spring Harbor Protocols
Cold Spring Harb Protoc 2006. 2006: pdb.prot4493-
Acridine Orange/Ethidium Bromide (AO/EB)
Staining to Detect Apoptosis
This protocol was adapted from “Apoptosis Assays,” Chapter 15, in Cells (eds. Spector et
al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. This
three-volume set is now out of print; however, some of the microscopy methods were
republished in Basic Methods in Microscopy, by David L. Spector and Robert D.
Shailaja Kasibhatla, , Gustavo P. Amarante-Mendes
Thomas Brunner, , Ella Bossy-Wetzel
Gustavo P. Amarante-Mendes, , Deborah Finucane
Ella Bossy-Wetzel and
Deborah Finucane, ,
and Douglas R. GreenDouglas R. Green
Acridine orange/ethidium bromide (AO/EB) staining is used to visualize nuclear
changes and apoptotic body formation that are characteristic of apoptosis. Cells
are viewed under a fluorescence microscope and counted to quantify apoptosis.
Fluorescence microscope with fluorescein filter and 60X objective
Slides and coverslips
1. Incubate 25 µl of cell suspension (0.5 × 106 to 2.0 × 106 cells/ml)
with 1 µl of AO/EB solution. Mix gently. Each sample should be mixed
just prior to microscopy and quantification. Samples must be
2. Place 10 µl of cell suspension onto a microscopic slide, cover with a
glass coverslip, and examine at least 300 cells in a fluorescence
microscope using a fluorescein filter and a 60X objective. (Higher or
lower magnification may be desired depending on cell type. Nuclear
morphology should be discernible.)
Acridine orange is a vital dye and will stain both live and dead cells.
Ethidium bromide will stain only cells that have lost membrane
integrity. Live cells will appear uniformly green. Early apoptotic cells
will stain green and contain bright green dots in the nuclei as a
consequence of chromatin condensation and nuclear fragmentation.
Late apoptotic cells will also incorporate ethidium bromide and
therefore stain orange, but, in contrast to necrotic cells, the late
apoptotic cells will show condensed and often fragmented nuclei.
Necrotic cells stain orange, but have a nuclear morphology
resembling that of viable cells, with no condensed chromatin.