Publication protocol
To determine the number of necrotic or apoptotic cells induced by the addition of LSL, cells were stained in situ with 10mg/ml acridine orange (Sigma-Aldrich Company Ltd, Dorset, UK) and1mg/ml ethidium bromide (Sigma-Aldrich Company Ltd, Dorset, UK) and morphological changes were assessed by fluorescence microscopy [31]. For assessment of apoptosis, a total of3x104cells were seeded onto a 10mm coverslip (Agar Scientific; Stansted, Essex, UK) and incubated overnight to form a confluent monolayer. 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 were incubated for a further 24h. To determine the number of live cells remaining on the coverslip the samples were washed three times with ice-cold phosphate-buffered 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 with ice-cold PBS and subsequent imaging with a Zeiss fluorescence microscope (Axio Scope 1, Zeiss, Germany) at a range of objective magnifications. The operator was blinded to the experimental groups and random fields were selected (40X objective). A total of 300 attached cells per cover-slip were 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).
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