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.

MATERIALS

Reagents

Cell suspension

AO/EB solution

Equipment

Fluorescence microscope with fluorescein filter and 60X objective

Slides and coverslips

METHOD

1. Incubate 25 µl of cell suspension (0.5 × 10

to 2.0 × 10

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
evaluated immediately.

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.