Acridine orange/ethidium bromide (AO/EtBr) double staining

The protocol has been provided below. If a more detailed protocol is required then the authors can contact us via e-mail. 

Protocol for Acrydine Orange/Ethidium Bromide double staining:
Acryridine Orange/Ethidium Bromide (AO/EtBr) Dual staining technique. In our study, cells were seeded in a 96-well plate at a density of approximately 10⁴ cells/well. Following incubation for 48 hours, cells were trypsinized and 10-25 μl of cell suspension was transferred onto glass microscope slides. 1 μl of AO/EtBr staining solution (a mixture of dyes containing 100 μg/ml AO and 100 μg/ml EtBr) was added on cell suspensions and then the samples were covered with a coverslip. The morphology of cells was examined under a fluorescent microscope (Carl-Zeiss/Axio observer 3., Zen 2.3 Blue Edition software) within 20 minutes after adding Ao/EtBr stain. For an accurate statistical analysis st least 200 cells should be counted and the results can be expressed as mean values obtained from at least three independent experiments. Both live and dead cells are stained with AO while, ethidium bromide stains only dead cells which have lost membrane integrity. Live cells appear uniformly green whereas early apoptotic cells show green dots in their nuclei. Late apoptotic cells stain orange and show condensed and/or often fragmented nuclei. Necrotic cells stain orange, with a nuclear morphology resembling that of viable cells, but without condensed chromatin.
 

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