Acridine Orange solution

Protocol tips

Upstream tips	Protocol tips	Downstream tips
• Dual Staining Procedure for differentiating Live/ Apoptotoic/Necrotic cells	• Seed 3x10^4 cells onto a 10mm coverslip and incubate overnight to form a confluent monolayer	• The operator to be blinded to the experimental groups and random fields to be selected (40X objective). • A total of 300 attached cells per cover-slip can be morphologically identified and counted as being either necrotic (red/orange nuclei),apoptotic (green condensed or fragmented nuclei) green or live (green non-condensed ovoid or rounded nuclei)

Upstream tips
• Dual Staining Procedure for differentiating Live/ Apoptotoic/Necrotic cells
Protocol tips
• Seed 3x10^4 cells onto a 10mm coverslip and incubate overnight to form a confluent monolayer
Downstream tips
• The operator to be blinded to the experimental groups and random fields to be selected (40X objective). • A total of 300 attached cells per cover-slip can be morphologically identified and counted as being either necrotic (red/orange nuclei),apoptotic (green condensed or fragmented nuclei) green or live (green non-condensed ovoid or rounded nuclei)

Publication protocol

Following serum starvation for 24h, LSL(20μg/ml or 70μg/ml) or 5μM of etoposide (control) (Sigma-Aldrich Company Ltd, Dorset,UK) was added and the cells incubated for a further 24h. To determine the number of live cellsremaining on the coverslip the samples were washed three times with ice-cold phosphate buff-ered saline (PBS; pH7.4, Oxoid: UK) 3 times, followed by incubation with a solution of 10μlof1:1 acridine orange/ethidium bromide for 5 minutes and then the cells were washed 3x withice-cold PBS and subsequent imaging with a Zeiss florescence microscope

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Manufacturer protocol

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